Intelligent CAR-T cell culture system

Abstract

The invention relates to the technical field of biological medicine preparation equipment, and discloses an intelligent CAR-T cell culture system, which comprises an intelligent temperature control and cleaning box body, a double sterilization system and a full-automatic preparation process, and the intelligent temperature control and cleaning box body adopts temperature and humidity control and cleanliness control. The double sterilization system comprises hydrogen peroxide fumigation and ultraviolet circulation disinfection, and the full-automatic preparation process comprises a precise pipeline, a modular reaction unit and an automatic control platform. According to the intelligent CAR-T cell culture system, full-process automatic culture of full-automatic CAR-T cells is realized in a C-level environment, a high-requirement clean environment is not needed, the cost can be greatly reduced, the economic benefit is remarkable, a full-closed pipeline system better meets the requirements of laws and regulations, and the system has greater advantages in the supervision level; the full-automatic operation reduces the intervention of personnel, avoids the potential pollution risk in the process, the full-process automatic cultivation can greatly save the cost, and the cost is saved by 40% compared with the traditional process through comprehensive calculation.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceutical preparation equipment, and specifically to an intelligent CAR-T cell cultivation system. Background Art

[0002] The intelligent CAR-T cell cultivation system is an advanced treatment method developed based on CAR-T cell therapy. It integrates multiple technologies such as cell culture, gene editing, automation and robotics, data analysis and artificial intelligence, aiming to improve the preparation efficiency and treatment effect of CAR-T cells. With the continuous advancement of technology and the expansion of application fields, intelligent CAR-T cell cultivation systems are expected to bring more hope and good news to cancer patients. The characteristics of the existing technology are generally to cultivate CAR-T cells by manual operation. The preparation process needs to be divided into multiple steps in the Class A and Class C environment of the GMP workshop, and processed step by step by single machines and manual labor. During each step of the process, an exposure will be formed when the cells are transferred, exposing the cells to the air, increasing the risk of bacterial contamination, leading to cultivation failure. Once the failure occurs, it will seriously affect the patient's treatment and endanger their lives. The existing technology requires the construction of Class A and Class C sterile and dust-free clean workshops. The construction cost, operation and maintenance cost are extremely high. The existing technology uses many single-machine equipment to complete the preparation of CAR-T cells. The equipment procurement cost is high, and the main equipment and consumables are all imported from abroad. Since the existing technology only provides a local clean environment, it still needs to integrate multiple single-machine equipment, which is costly and inefficient. The degree of automation is low, and it is usually carried out by robot plus manual assistance. The existence of exposure cannot be avoided during the cell transfer process, and there is a risk of exposure and contamination. There are challenges at the regulatory level. Therefore, this application now proposes an intelligent CAR-T cell cultivation system. Summary of the Invention

[0003] (1) Technical problems solved In response to the shortcomings of existing technologies, the present invention provides an intelligent CAR-T cell cultivation system that requires no human intervention from cell separation to finished product. It combines dual sterilization with VHP and UV-C to reduce contamination risks, enable flexible production, and enhance data traceability and quality control. This solves the problem that traditional CAR-T preparation relies on manual operation, dispersed equipment, and high costs, resulting in high contamination risks, long production cycles, and limited indications. There is an urgent need to reduce costs and increase efficiency through automated integrated equipment and innovative technologies.

[0004] (2) Technical solution To achieve the above objectives, the present invention provides the following technical solutions: an intelligent CAR-T cell cultivation system, comprising an intelligent temperature control and clean box, a dual sterilization system, and a fully automated preparation process. The intelligent temperature control and clean box utilizes temperature and humidity control and cleanliness control. The dual sterilization system includes hydrogen peroxide fumigation and ultraviolet cyclic disinfection. The fully automated preparation process includes precision piping, modular reaction units, and an automated control platform. The operating steps of the intelligent CAR-T cell cultivation system are as follows: S1, first 200ml single blood → centrifuge bottle, then 20ml sorting magnetic beads → centrifuge bottle; S2. Centrifuge and incubate, place 200 ml of the cell from the centrifuge bottle into the cell bag, and then from the cell bag → sorting column → waste bag; S3, 110ml sorting solution → sorting column → centrifuge bottle, 50ml culture medium solution → centrifuge bottle → waste bag; S4, centrifuge bottle → 5ml sampling bag 1, 100ml freezing solution → centrifuge bottle → aliquot → cryopreservation; S5, recovery → centrifuge bottle, recover the frozen cells, transfer the liquid to the centrifuge bottle, 100 ml of culture medium solution → centrifuge bottle → waste bag, wash, drain the waste liquid to the waste bag, repeat the step 3-5 times; S6, 40ml activated magnetic beads → centrifuge bottle, rotate the centrifuge bottle, start incubation, centrifuge incubation for 30 minutes, after incubation, transfer to cell culture bag 1; S7. Add appropriate culture medium solution to cell culture bag 1, incubate for 24 hours to activate T cells, transfer the solution to a centrifuge bottle, centrifuge, leave the activated T cells in the bottle, and drain the waste liquid into a waste bag; S8, 20 ml virus → centrifuge bottle, centrifuge bottle → cell culture bag 1, culture medium → cell culture bag 1, add appropriate culture medium solution to cell culture bag 1, and culture cells statically for 24 hours; S9, cell culture bag 1 → magnet → cell culture bag 2. During the process of transferring liquid from cell culture bag 1 to cell culture bag 2, the liquid passes through the magnet to remove the activated magnetic beads. S10, cell culture bag 2 → 5ml sampling bag 2, count, measure cell density, observe cell number, culture medium → cell culture bag 2 → waste bag, add appropriate culture medium, dynamic culture, after 2-3 days, start perfusion, sample and observe every day, after 4-5 days, prepare for harvest; S11, cell culture bag 2 → centrifuge bottle → waste bag, after the culture is completed, the cell suspension is transferred to the centrifuge bottle, centrifuged, leaving the cultured T cells in the bottle, and the waste liquid is discharged to the waste bag, 100ml sorting solution → centrifuge bottle → waste bag; S12, centrifuge bottle → 5ml sampling bag, count, measure cell density, observe cell number, freezing solution → centrifuge bottle → packaging, after counting the cultured cells, add appropriate freezing solution to the centrifuge and mix well, and package the finished product and samples.

[0005] Preferably, the temperature and humidity control uses high-precision sensors and a dynamic adjustment system. The temperature inside the box is kept constant at 37±0.5°C and the humidity is kept stable at 60±5%, ensuring optimal cell activity and culture conditions.

[0006] Preferably, the interior of the cleanliness control box is a Class 100 clean environment, equipped with a laminar air supply system and a HEPA high-efficiency filtration device to monitor the concentration of particulate matter in real time to prevent external pollution.

[0007] Preferably, the hydrogen peroxide fumigation evenly releases gaseous hydrogen peroxide through an atomizing device, deeply sterilizing the internal pipes, cavities and consumables of the box, killing spores and viruses, with a sterilization efficiency of 6-log level.

[0008] Preferably, the ultraviolet cycle disinfection automatically starts the UV-C ultraviolet lamp during the operation interval to perform surface disinfection on the exposed area to avoid cross contamination.

[0009] Preferably, the precision piping and modular reaction unit adopt a closed disposable piping system, integrating cell separation, activation, gene transduction, and amplification modules, achieving precise liquid delivery through solenoid valves and peristaltic pumps, and supporting single-batch or multi-batch parallel processing.

[0010] Preferably, the automated control platform is based on PLC and AI algorithms to monitor cell concentration and metabolic parameters in real time, dynamically adjust the culture medium replenishment rate and transduction time, and ensure process consistency.

[0011] Compared with the existing technology, the present invention provides an intelligent CAR-T cell cultivation system with the following beneficial effects: 1. This intelligent CAR-T cell cultivation system realizes the full process of fully automatic CAR-T cell cultivation in a Class C environment. It does not require a high-quality clean environment, which can significantly reduce costs and achieve significant economic benefits. During the process, strict control of cleanliness, temperature, humidity, light and other conditions is carried out to better provide a favorable environment for cell culture. The pipelines are fully enclosed and have no openings, ensuring that there is no contact with the outside air during the cultivation process, completely avoiding pollution and improving the success rate of cultivation. 2. The intelligent CAR-T cell cultivation system has a fully enclosed piping system that better complies with regulatory requirements and has greater advantages at the supervisory level. Fully automated operation reduces human intervention and avoids potential contamination risks during the process. Fully automated cultivation can significantly save costs, including consumables loss, production site occupancy, and labor costs. Comprehensive calculations show a 40% cost savings compared to traditional processes. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Schematic diagram of the cultivation system structure of the present invention; Figure 2 For the present invention Figure 1 Partial schematic diagram of the cultivation system; Figure 3 For the present invention Figure 1 Partial schematic diagram of the cultivation system; Figure 4 For the present invention Figure 1 Partial schematic diagram of the cultivation system. DETAILED DESCRIPTION

[0013] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0014] An intelligent CAR-T cell cultivation system includes an intelligent temperature control and cleanroom chamber, a dual sterilization system, and a fully automated preparation process. The intelligent temperature control and cleanroom chamber utilizes temperature, humidity, and cleanliness control. The dual sterilization system includes hydrogen peroxide fumigation and ultraviolet cyclic disinfection. The fully automated preparation process includes precision piping, modular reaction units, and an automated control platform. The operating steps of the intelligent CAR-T cell cultivation system are as follows: S1, first 200ml single blood → centrifuge bottle, then 20ml sorting magnetic beads → centrifuge bottle; S2. Centrifuge and incubate, place 200 ml of the cell from the centrifuge bottle into the cell bag, and then from the cell bag → sorting column → waste bag; S3, 110ml sorting solution → sorting column → centrifuge bottle, 50ml culture medium solution → centrifuge bottle → waste bag; S4, centrifuge bottle → 5ml sampling bag 1, 100ml freezing solution → centrifuge bottle → aliquot → cryopreservation; S5, recovery → centrifuge bottle, recover the frozen cells, transfer the liquid to the centrifuge bottle, 100 ml of culture medium solution → centrifuge bottle → waste bag, wash, drain the waste liquid to the waste bag, repeat the step 3-5 times; S6, 40ml activated magnetic beads → centrifuge bottle, rotate the centrifuge bottle, start incubation, centrifuge incubation for 30 minutes, after incubation, transfer to cell culture bag 1; S7. Add appropriate culture medium solution to cell culture bag 1, incubate for 24 hours to activate T cells, transfer the solution to a centrifuge bottle, centrifuge, leave the activated T cells in the bottle, and drain the waste liquid into a waste bag; S8, 20 ml virus → centrifuge bottle, centrifuge bottle → cell culture bag 1, culture medium → cell culture bag 1, add appropriate culture medium solution to cell culture bag 1, and culture cells statically for 24 hours; S9, cell culture bag 1 → magnet → cell culture bag 2. During the process of transferring liquid from cell culture bag 1 to cell culture bag 2, the liquid passes through the magnet to remove the activated magnetic beads. S10, cell culture bag 2 → 5ml sampling bag 2, count, measure cell density, observe cell number, culture medium → cell culture bag 2 → waste bag, add appropriate culture medium, dynamic culture, after 2-3 days, start perfusion, sample and observe every day, after 4-5 days, prepare for harvest; S11, cell culture bag 2 → centrifuge bottle → waste bag, after the culture is completed, the cell suspension is transferred to the centrifuge bottle, centrifuged, leaving the cultured T cells in the bottle, and the waste liquid is discharged to the waste bag, 100ml sorting solution → centrifuge bottle → waste bag; S12, centrifuge bottle → 5ml sampling bag, count, measure cell density, observe cell number, freezing solution → centrifuge bottle → packaging, after counting the cultured cells, add appropriate freezing solution to the centrifuge and mix well, and package the finished product and samples.

[0015] Furthermore, the temperature and humidity control uses high-precision sensors and a dynamic adjustment system. The temperature inside the box is constant at 37±0.5°C and the humidity is stable at 60±5%, ensuring optimal cell activity and culture conditions.

[0016] Furthermore, the interior of the cleanliness control box is a Class 100 clean environment, equipped with a laminar air supply system and a HEPA high-efficiency filtration device to monitor the concentration of particulate matter in real time to prevent external contamination.

[0017] Furthermore, the hydrogen peroxide fumigation evenly releases gaseous hydrogen peroxide through an atomization device, deeply sterilizing the internal pipes, cavities and consumables of the box, killing spores and viruses, with a sterilization efficiency of 6-log level.

[0018] Furthermore, the ultraviolet cycle disinfection automatically starts the UV-C lamp during operation to disinfect the surface of the exposed area to avoid cross contamination.

[0019] Furthermore, the precision piping and modular reaction unit adopt a closed disposable piping system, integrating cell separation, activation, gene transduction, and amplification modules, achieving precise liquid delivery through solenoid valves and peristaltic pumps, and supporting single-batch or multi-batch parallel processing.

[0020] Furthermore, the automated control platform is based on PLC and AI algorithms to monitor cell concentration and metabolic parameters in real time, dynamically adjust the culture medium replenishment rate and transduction time, and ensure process consistency.

[0021] The specific steps for CAR-T cell cultivation are as follows: 1.200ml single blood → centrifuge bottle: all finger pinch valves are closed, open finger pinch valves 1, 2, and 3, and when the single blood has completely entered the centrifuge bottle, close finger pinch valves 1 and 2. 2.20ml sorting magnetic beads → centrifuge bottle (the liquid flow meter measures 20ml of sorting magnetic beads. The amount of 20ml of sorting magnetic beads is too little to reach the centrifuge bottle. At this time, use the sorting solution to flush the sorting magnetic beads in the tube into the centrifuge bottle. Note: the sorting solution only flushes the sorting magnetic beads into the centrifuge bottle. When 20ml of sorting magnetic beads enter the centrifuge bottle, the addition of sorting solution should be stopped immediately. A small amount of sorting solution is allowed in the centrifuge bottle): liquid flow meter No. 1 is turned on, liquid flow meter No. 1 is reset to zero, finger pinch valves 4 and 5 are opened, and the sorting magnetic beads pass through liquid flow meter No. 1. When 20ml of sorting magnetic beads are measured, finger pinch valve No. 4 is closed, and finger pinch valve No. 6 is opened. The sorting solution flushes the sorting magnetic beads in the tube into the centrifuge bottle. When the sorting magnetic beads have completely entered the centrifuge bottle, finger pinch valves 6, 5, and 3 are closed.

[0022] 3. Centrifugal incubation (the centrifuge bottle rotates and incubation begins. The centrifugal incubation lasts for 30 minutes. At this time, the bottle contains 200 ml of single-sample blood, 20 ml of sorting magnetic beads and a small amount of sorting solution. After incubation, the bottle contains a single-sample blood sorting magnetic bead suspension): the centrifuge bottle starts to rotate and incubation begins. The speed is rpm (the motor has a maximum speed of 6000 rpm and is adjustable). The incubation time is 30 minutes. After the incubation is completed, the centrifuge bottle stops rotating.

[0023] 4. Centrifuge bottle → cell bag: Open finger pinch valves 3, 2, and 7, and rotate peristaltic pump 1 clockwise to pipette the single blood sorting magnetic bead suspension into the cell bag. When the single blood sorting magnetic bead suspension has completely entered the cell bag, close finger pinch valves 3 and 2 and stop peristaltic pump 1.

[0024] 5. Cell bag → Sorting column → Waste bag (50ml of single blood collection magnetic bead suspension enters the sorting column, which can filter out cell clusters and impurities such as red blood cells and platelets other than T cells in the blood. These impurities are finally discharged into the waste bag. At this time, the sorting column contains T cells labeled with sorting magnetic beads): The electromagnet turns on, liquid flow meter No. 2 turns on, liquid flow meter No. 2 is reset, finger pinch valve No. 11 is opened, peristaltic pump No. 1 rotates counterclockwise, and the single blood collection magnetic bead suspension passes through liquid flow meter No. 2. When 50ml of single blood collection magnetic bead suspension is measured, peristaltic pump No. 1 stops.

[0025] 6.110ml sorting solution → sorting column → centrifuge bottle (110ml sorting solution flushes the sorted magnetic bead-labeled T cells in the sorting column into the centrifuge bottle): Clear liquid flow meter No. 2, open finger pinch valve No. 8, and allow the sorting solution to flow through liquid flow meter No. 2. When 10ml of sorting solution is measured, finger pinch valves No. 7 and No. 11 close, turn off the electromagnet, clear liquid flow meter No. 2, open finger pinch valves No. 9, No. 10, No. 2, and No. 3, and allow the sorting solution to flow through liquid flow meter No. 2. When 100ml of sorting solution is measured, finger pinch valves No. 8, No. 9, No. 10, and No. 2 close.

[0026] 7.50ml culture medium solution → centrifuge bottle → waste bag (wash, leave T cells labeled with sorting magnetic beads in the bottle, drain the waste liquid into the waste bag, repeat steps 5, 6, and 7 until all the apheresis magnetic bead suspension in the cell bag is sorted, continue to repeat step 7 2~3 times, leaving T cells labeled with sorting magnetic beads in the bottle): reset the No. 1 liquid flow meter, open the finger pinch valve 18, No. 5, and let the culture medium solution pass through the No. 1 liquid flow meter. When 50ml of culture medium solution is measured, open the finger pinch valve 18, No. 5, No. 3 Close, the centrifuge bottle starts to rotate and starts washing (the motor has a maximum speed of 6000 rpm and the speed is adjustable). After washing is completed, the centrifuge bottle stops, and the finger pinch valves 3, 2, and 11 are opened. The peristaltic pump 2 rotates counterclockwise to discharge the waste liquid in the centrifuge bottle into the waste bag. When the light sensor / camera in the bottle senses the cells at the bottle mouth, the peristaltic pump 2 stops rotating, and the finger pinch valves 3, 2, and 11 are closed. Repeat steps 5, 6, and 7 until all the single blood sorting magnetic bead suspension in the cell bag is sorted. Repeat step 7 2 to 3 times.

[0027] 8. Centrifuge bottle → 5ml sampling bag 1 (count, measure cell density, and observe cell number. Note: If you do not need to freeze cells, skip steps 9, 10, and 11 and proceed directly to step 12): Open finger pinch valves 12 and 13, turn peristaltic pump 3 clockwise, and pump 5ml of washed cells into the sampling bag at Luer interface 1. Stop peristaltic pump 3 and close finger pinch valves 12 and 13.

[0028] 9. Appropriate (100ml) cryopreservation solution → centrifuge bottle → aliquot → cryopreservation (After washing and counting the cells, add appropriate cryopreservation solution to the centrifuge, mix thoroughly, aliquot, and cryopreservation): Open finger pinch valves 12, 14, and 15, rotate peristaltic pump 3 counterclockwise, and transfer the cryopreservation solution from the Luer ports 2 and 3 to the centrifuge bottle. Stop peristaltic pump 3, close finger pinch valves 15, 14, and 12, and start rotating the centrifuge bottle to mix thoroughly (motor speed is adjustable, up to 6000 rpm). Once mixing is complete, stop the centrifuge bottle. Open finger pinch valves 12, 16, and 17, and rotate peristaltic pump 3 clockwise until all the mixed solution in the centrifuge bottle is transferred to the aliquot bag with the Luer ports 4 and 5. Once the aliquot is transferred, stop peristaltic pump 3, and close finger pinch valves 12, 16, and 17. (Aliquots are cryopreserved) 10 Recovery → Centrifuge bottle (Resuscitate the frozen cells and transfer them to the centrifuge bottle): (Resuscitate the frozen cells and transfer them to the Luer ports 4 and 5) Open finger pinch valves 14 and 15, and rotate peristaltic pump 3 counterclockwise until all the cell solution in the Luer ports 4 and 5 is transferred to the centrifuge bottle. Stop peristaltic pump 3 and close finger pinch valves 12, 16, and 17.

[0029] 11. 100ml culture medium solution → centrifuge bottle → waste bag (wash, drain waste liquid to waste bag, repeat step 11, 3-5 times): Clear liquid flow meter No. 1, open finger pinch valves 18, 5, and 3, and let the culture medium solution pass through liquid flow meter No. 1. When 100ml of culture medium solution is measured, finger pinch valves 18, 5, and 3 close, and the centrifuge bottle starts to rotate and wash begins (motor speed is up to 6000 rpm, adjustable speed). When wash is complete, the centrifuge bottle stops rotating, open finger pinch valves 3, 2, and 11, and peristaltic pump No. 2 rotates counterclockwise to drain the waste liquid in the centrifuge bottle to the waste bag. When the light sensor / camera in the bottle senses that the cells have reached the bottle mouth, peristaltic pump No. 2 stops rotating, and finger pinch valves 3, 2, and 11 close. Repeat steps 11, 3-5 times.

[0030] 12.40ml activated magnetic beads → centrifuge bottle (40ml activated magnetic beads are too small to enter the centrifuge bottle completely. At this time, use culture medium to flush the activated magnetic beads in the tube into the centrifuge bottle. Note: The culture medium is only used to flush the activated magnetic beads into the centrifuge bottle. When 40ml of activated magnetic beads enter the centrifuge bottle, the addition of culture medium should be stopped immediately. A small amount of culture medium is allowed in the centrifuge bottle. Note: The cytokines added to the culture medium should be mixed in advance. This culture medium solution is also added to the cell culture bag 1 later. The shelf life of the mixed culture medium solution is 48H / 72H): Liquid flow meter No. 1 is reset to zero, finger pinch valves 19, 5, and 3 are opened, and the activated magnetic beads pass through liquid flow meter No. 1. When 45ml of activated magnetic beads are measured, finger pinch valve No. 19 is closed, and finger pinch valve No. 18 is opened. The culture medium solution flushes the activated magnetic beads in the tube into the centrifuge bottle. When the activated magnetic beads have completely entered the centrifuge bottle, finger pinch valves 18, 5, and 3 are closed.

[0031] 13. Centrifugal incubation (centrifuge bottle rotates, incubation starts, centrifugal incubation for 30 minutes): The centrifuge bottle starts to rotate and incubation begins (the motor has a maximum speed of 6000 rpm and the speed is adjustable). The incubation time is 30 minutes. When the incubation is completed, the centrifuge bottle stops rotating.

[0032] 14. Centrifuge bottle → Cell culture bag 1 (after incubation, transfer to cell culture bag 1): Rotate the pinch valve to place the static incubator horizontally. Open finger pinch valves 3, 2, 10, 20, and 21. Turn peristaltic pump 4 clockwise until all the activated magnetic bead T cell mixture in the centrifuge bottle is transferred to cell culture bag 1. Stop peristaltic pump 4 and close finger pinch valves 3, 2, 10, and 20.

[0033] 15. Culture medium → Cell culture bag 1 (add appropriate culture medium solution to cell culture bag 1 and incubate for 24 hours to activate T cells): Open finger pinch valve 22, rotate peristaltic pump 4 clockwise, transfer culture medium solution to centrifuge bottle, stop peristaltic pump 4, close finger pinch valves 21 and 22, heat static cell culture plate to 37°C, and after the static incubator reaches 37°C, use micro-infrared gas sensor 1 to sense the carbon dioxide concentration of 5% in the static incubator. Incubate at a constant temperature of 37°C. Open finger pinch valve 23, rotate peristaltic pump 5 clockwise, and after the liquid sensor senses that liquid has entered the sink, stop peristaltic pump 5, close finger pinch valve 23, and incubate for 24 hours.

[0034] 16. Cell culture bag 1 → centrifuge bottle → waste bag (after static culture, transfer the liquid to the centrifuge bottle, centrifuge, leave the activated T cells in the bottle, and drain the waste liquid to the waste bag): After 24 hours, stop heating the static cell culture plate, open finger pinch valve No. 23, and rotate peristaltic pump No. 5 counterclockwise until all the water in the sink is sucked out. Rotate the pinch valve to place the static incubator vertically, stop peristaltic pump No. 5, close finger pinch valve No. 23, open finger pinch valves No. 3, 2, 10, 20, and 21, and rotate peristaltic pump No. 4 counterclockwise. The solution is transferred to the centrifuge bottle, peristaltic pump No. 4 stops, finger pinch valves 3, 2, 10, 20, and 21 are closed, the centrifuge bottle starts to rotate, and washing begins (the motor has a maximum speed of 6000 rpm and the speed is adjustable). After washing is completed, the centrifuge bottle stops, finger pinch valves 3, 2, and 11 are opened, and peristaltic pump No. 2 rotates counterclockwise to drain the waste liquid in the centrifuge bottle into the waste bag. When the light sensor / camera in the bottle senses that the cells have reached the bottle mouth, peristaltic pump No. 2 stops, finger pinch valves 3, 2, and 11 are closed, and step 16 is repeated until all the activated cell solution is washed.

[0035] 17. 20ml virus → centrifuge bottle (Add 20ml of virus to the centrifuge bottle for transduction. 20ml of virus is too little to reach the centrifuge bottle. In this case, use culture medium solution to flush the virus in the tube into the centrifuge bottle. Note: The culture medium solution only flushes the virus into the centrifuge bottle. Once 20ml of virus has entered the centrifuge bottle, stop adding culture medium solution immediately. A small amount of culture medium solution is allowed in the centrifuge bottle. Reset liquid flow meter No. 1, open finger pinch valves 24 and 5, and allow the virus to pass through liquid flow meter No. 1. When 20ml of virus has been measured, close finger pinch valve No. 24 and open finger pinch valve No. 25. The culture medium solution flushes the virus in the tube into the centrifuge bottle. When the virus has completely entered the centrifuge bottle, close finger pinch valves 25, 5, and 3.

[0036] 18. Centrifuge bottle → Cell culture bag 1 (after transduction, transfer to cell culture bag 1): Rotate the pinch valves to position the static incubator horizontally. Open finger pinch valves 3, 2, 10, 20, and 21. Turn peristaltic pump 4 clockwise until all the virus-transduced T cell solution in the centrifuge bottle is transferred to cell culture bag 1. Stop peristaltic pump 4 and close finger pinch valves 3, 2, 10, and 20.

[0037] 19. Culture medium → Cell culture bag 1 (add appropriate culture medium solution to cell culture bag 1 and culture cells statically for 24 hours): Open finger pinch valve 22, rotate peristaltic pump 4 clockwise, transfer culture medium solution to centrifuge bottle, stop peristaltic pump 4, close finger pinch valves 21 and 22, heat static cell culture plate to 37°C, and after the static incubator reaches 37°C, use micro-infrared gas sensor 1 to sense the carbon dioxide concentration of 5% in the static incubator. Incubate at a constant temperature of 37°C. Open finger pinch valve 23, rotate peristaltic pump 5 clockwise, and after the liquid sensor senses that liquid has entered the sink, stop peristaltic pump 5, close finger pinch valve 23, and culture statically for 24 hours.

[0038] 20. Cell culture bag 1 → magnet → cell culture bag 2 (cell culture bag 1 passes through the magnet during transfer to cell culture bag 2 to remove activated magnetic beads): After 24 hours, stop heating the static cell culture plate, open finger pinch valve No. 23, and rotate peristaltic pump No. 5 counterclockwise until all the water in the sink is drawn out. Rotate the pinch valve to place the static incubator vertically, stop peristaltic pump No. 5, close finger pinch valve No. 23, open finger pinch valves No. 26 and No. 27, and rotate peristaltic pump No. 4 counterclockwise until all the T cell solution cultured after virus transduction is transferred to cell culture bag 2. Stop peristaltic pump No. 4, and close finger pinch valves No. 26 and No. 27.

[0039] 21. Cell culture bag 2 → 5 ml sampling bag 2 (counting, measuring cell density, and observing cell number): Open finger pinch valve 28 and turn peristaltic pump 6 clockwise to pump 5 ml of the virus-transduced cell solution into the sampling bag at Luer interface 10. Stop peristaltic pump 6 and close finger pinch valve 28.

[0040] 22. Culture medium → Cell culture bag 2 → Waste bag (add appropriate culture medium, culture dynamically, start perfusion after 2-3 days, take samples every day and observe, prepare for harvest after 4-5 days. Perfusion: replace the culture medium solution, and the old culture medium solution flows into the waste bag. Note: The culture medium used in cell culture bag 2 needs to be added with 2 times the amount of cytokine ratio in cell culture bag 1. Pay attention to the ratio time. The shelf life of the well-proportioned culture medium solution is 48 hours / 72 hours): Open finger pinch valves 22, 28, and 29, and peristaltic pump. The dynamic pump No. 6 rotates counterclockwise, and the peristaltic pump No. 6 rotates to take the culture medium solution to the cell culture bag 2. The peristaltic pump No. 6 stops rotating, and the finger pinch valves 22, 28, and 29 are closed. The dynamic cell culture plate starts to shake (the motor has a maximum speed of 6000 rpm, the speed is adjustable, and the shaking angle is adjustable) and heats to 37°. The valve of the carbon dioxide gas tank is opened, and the micro infrared gas sensor 2 senses the carbon dioxide concentration in the trachea to be 5%. After the dynamic incubator reaches 37°, the cells are cultured at a constant temperature of 37° for 48 to 72 hours. After 48 to 72 hours, perfuse and take samples every day for observation, and cultivate for 4 to 5 days (open finger pinch valves 22, 29, 28, and 30, rotate peristaltic pumps 2 and 6 counterclockwise to start perfusion, stop peristaltic pumps 2 and 6, close finger pinch valves 22, 29, 28, and 30, after 24 hours, open finger pinch valve 28, rotate peristaltic pump 6 clockwise, rotate peristaltic pump 6, and peristaltically pump 5 ml of the incubated cell solution into the sampling bag of Luer interface 10, stop peristaltic pump 6, close finger pinch valve 28, and repeat 4 to 5 times).

[0041] 23. Cell culture bag 2 → centrifuge bottle → waste bag (After the cell culture is completed, the cell suspension is transferred to the centrifuge bottle and centrifuged, leaving the cultured T cells in the bottle, and the waste liquid is discharged to the waste bag): After the cell culture is completed, the dynamic cell culture plate stops shaking and heating, the CO2 gas tank valve is closed, finger pinch valves 3, 2, 10, 20, 29, and 28 are opened, and peristaltic pump 6 rotates clockwise. The cultured cell solution is transferred to the centrifuge bottle, the peristaltic pump 6 stops rotating, and finger pinch valve 3 is opened. , 2, 10, 20, 29, 28 are closed, the centrifuge bottle is turned on and rotated, and washing begins (the motor has a maximum speed of 6000 rpm and the speed is adjustable). After washing is completed, the centrifuge bottle stops rotating, and the finger pinch valves 3, 2, and 11 are opened. The peristaltic pump No. 2 rotates counterclockwise to discharge the waste liquid in the centrifuge bottle into the waste bag. When the light sensor / camera in the bottle senses the cells at the bottle mouth, the peristaltic pump No. 2 stops rotating, and the finger pinch valves 3, 2, and 11 are closed. Repeat step 23 until all the cultured cell solutions are washed.

[0042] 24. 100ml sorting solution → centrifuge bottle → waste bag (wash, drain waste liquid to waste bag, repeat steps 23, 3-5 times): Clear liquid flow meter No. 1, open finger pinch valves 6, 5, and 3, and allow sorting solution to flow through liquid flow meter No. 1. When 100ml of sorting solution is measured, finger pinch valves 6, 5, and 3 close, and the centrifuge bottle starts rotating to begin washing (motor speed is adjustable, up to 6000 rpm). Washing is completed for 3 minutes, and the centrifuge bottle stops rotating. Open finger pinch valves 3, 2, and 11, and peristaltic pump No. 2 rotates counterclockwise to drain the waste liquid in the centrifuge bottle to the waste bag. When the light sensor / camera in the bottle senses that the cells have reached the bottle mouth, peristaltic pump No. 2 stops rotating, and finger pinch valves 3, 2, and 11 close. Repeat steps 24, 3-5 times.

[0043] 25. Centrifuge bottle → 5ml sampling bag (counting, measuring cell density, observing cell number): Open finger pinch valves 12 and 13, turn peristaltic pump 3 clockwise, and pump 5ml of washed cells into the sampling bag at Luer interface 1. Stop peristaltic pump 3 and close finger pinch valves 12 and 13.

[0044] 26. Cryopreservative → Centrifuge Bottle → Aliquoting (After counting cultured cells, add appropriate cryopreservative solution to the centrifuge for mixing, and then aliquot the finished product and samples): Open finger pinch valves 12, 14, and 15, and rotate peristaltic pump 3 counterclockwise. Transfer cryopreservative solution from Luer ports 2 and 3 to the centrifuge bottle. Stop peristaltic pump 3, close finger pinch valves 15, 14, and 12, and start rotating the centrifuge bottle to mix (motor speed adjustable, up to 6000 rpm). Once mixing is complete, stop rotating the centrifuge bottle. Open finger pinch valves 12, 31, 32, 33, and 34, and rotate peristaltic pump 3 clockwise until all the mixed solution in the centrifuge bottle is transferred to the aliquot bag with Luer ports 6, 7, 8, and 9. Once transfer is complete, stop peristaltic pump 3, and close finger pinch valves 12, 31, 32, 33, and 34.

[0045] The fully automated culture of CAR-T cells throughout the entire process can be achieved in a Class C environment. This does not require a high-level clean environment, can significantly reduce costs, and has significant economic benefits. Cleanliness, temperature, humidity, light and other conditions are strictly controlled during the process to provide a better environment for cell culture. The pipelines are fully enclosed and have no openings, ensuring that they are not exposed to the outside air during the culture process, completely avoiding pollution and improving the success rate of culture. The fully enclosed piping system is more in line with regulatory requirements and has greater advantages at the regulatory level. Fully automated operation reduces human intervention and avoids potential contamination risks in the process. Fully automated culture can significantly save costs, including consumables loss, production site occupancy, and labor in multiple dimensions. Comprehensive calculations show a 40% cost savings compared to traditional processes. It is suitable for the preparation of CAR-T cells in hospitals and biopharmaceutical companies, especially for scenarios that require rapid response and high cleanliness requirements during treatment.

[0046] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. An intelligent CAR-T cell cultivation system, characterized by: It includes an intelligent temperature control and clean box, a dual sterilization system, and a fully automated preparation process. The intelligent temperature control and clean box adopts temperature, humidity and cleanliness control. The dual sterilization system includes hydrogen peroxide fumigation and ultraviolet cycle disinfection. The fully automated preparation process includes precision piping, modular reaction units, and an automated control platform. The working steps of the intelligent CAR-T cell cultivation system are as follows: S1, first 200ml single blood → centrifuge bottle, then 20ml sorting magnetic beads → centrifuge bottle; S2. Centrifuge and incubate, place 200 ml of the cell from the centrifuge bottle into the cell bag, and then from the cell bag → sorting column → waste bag; S3, 110ml sorting solution → sorting column → centrifuge bottle, 50ml culture medium solution → centrifuge bottle → waste bag; S4, centrifuge bottle → 5ml sampling bag 1, 100ml freezing solution → centrifuge bottle → aliquot → cryopreservation; S5, recovery → centrifuge bottle, recover the frozen cells, transfer the liquid to the centrifuge bottle, 100 ml of culture medium solution → centrifuge bottle → waste bag, wash, drain the waste liquid to the waste bag, repeat the step 3-5 times; S6, 40ml activated magnetic beads → centrifuge bottle, rotate the centrifuge bottle, start incubation, centrifuge incubation for 30 minutes, after incubation, transfer to cell culture bag 1; S7. Add appropriate culture medium solution to cell culture bag 1, incubate for 24 hours to activate T cells, transfer the solution to a centrifuge bottle, centrifuge, leave the activated T cells in the bottle, and drain the waste liquid into a waste bag; S8, 20 ml virus → centrifuge bottle, centrifuge bottle → cell culture bag 1, culture medium → cell culture bag 1, add appropriate culture medium solution to cell culture bag 1, and culture cells statically for 24 hours; S9, cell culture bag 1 → magnet → cell culture bag 2. During the process of transferring liquid from cell culture bag 1 to cell culture bag 2, the liquid passes through the magnet to remove the activated magnetic beads. S10, cell culture bag 2 → 5ml sampling bag 2, count, measure cell density, observe cell number, culture medium → cell culture bag 2 → waste bag, add appropriate culture medium, dynamic culture, after 2-3 days, start perfusion, sample and observe every day, after 4-5 days, prepare for harvest; S11, cell culture bag 2 → centrifuge bottle → waste bag, after the culture is completed, the cell suspension is transferred to the centrifuge bottle, centrifuged, leaving the cultured T cells in the bottle, and the waste liquid is discharged to the waste bag, 100ml sorting solution → centrifuge bottle → waste bag; S12, centrifuge bottle → 5ml sampling bag, count, measure cell density, observe cell number, freezing solution → centrifuge bottle → packaging, after counting the cultured cells, add appropriate freezing solution to the centrifuge and mix well, and package the finished product and samples.

2. The intelligent CAR-T cell cultivation system according to claim 1, characterized in that: The temperature and humidity control uses high-precision sensors and a dynamic adjustment system. The temperature inside the box is kept constant at 37±0.5°C and the humidity is stabilized at 60±5%, ensuring optimal cell activity and culture conditions.

3. The intelligent CAR-T cell cultivation system according to claim 1, characterized in that: The interior of the cleanliness control box is a Class 100 clean environment, equipped with a laminar air supply system and HEPA high-efficiency filtration device to monitor particulate matter concentration in real time to prevent external contamination.

4. The intelligent CAR-T cell cultivation system according to claim 1, characterized in that: The hydrogen peroxide fumigation evenly releases gaseous hydrogen peroxide through an atomization device, deeply sterilizing the internal pipes, cavities and consumables of the box, killing spores and viruses, with a sterilization efficiency of 6-log level.

5. The intelligent CAR-T cell cultivation system according to claim 1, characterized in that: The UV cycle disinfection automatically starts the UV-C lamp during operation to disinfect the surface of the exposed area to avoid cross contamination.

6. The intelligent CAR-T cell cultivation system according to claim 1, characterized in that: The precision piping and modular reaction unit adopts a closed disposable piping system, integrating cell separation, activation, gene transduction, and amplification modules, and realizes precise liquid delivery through solenoid valves and peristaltic pumps, supporting single batch or multi-batch parallel processing.

7. The intelligent CAR-T cell cultivation system according to claim 1, characterized in that: The automated control platform is based on PLC and AI algorithms to monitor cell concentration and metabolic parameters in real time, dynamically adjust the culture medium replenishment rate and transduction time, and ensure process consistency.

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