A method for rapidly preparing CAR-T cells based on a closed loop microfluidic and application thereof

Abstract

The application belongs to the technical field of immunology and biological medicine, and particularly relates to a method for rapidly preparing CAR-T cells based on a closed-loop microfluidic system and application thereof. The closed-loop microfluidic system provided by the application comprises a liquid storage device, a connecting pipeline, a circulating driving unit, a bubble buffer / capture unit, a microfluidic chip, a liquid return pipeline and a temperature control culture module, and the liquid storage device, the circulating driving unit, the bubble buffer / capture unit, the microfluidic chip and the liquid return pipeline constitute a closed circulation loop. After mixing the short-time activated T cells with low MOI viruses, the application introduces the mixture into the closed-loop microfluidic system, so that the mixture repeatedly passes through a shallow wide channel type limited transduction channel in the closed loop to obtain CAR-T cells. The application improves the CAR positive rate and virus utilization rate under the condition of low MOI, significantly shortens the preparation period, maintains high cell survival rate, recovery rate and in-vitro anti-tumor function, reduces the virus dosage and the manufacturing cost, and is suitable for the clinical demand of rapid drug administration and automatic closed manufacturing of CAR-T cells.

Description

Technical Field

[0001] This invention belongs to the fields of immunology and biomedicine, specifically relating to a method for rapid preparation of CAR-T cells based on closed-loop microfluidics and its application. Background Technology

[0002] CAR-T cell (chimeric antigen receptor T cell) therapy has achieved significant clinical efficacy in hematological malignancies. However, the conventional preparation process involves multiple steps, including T cell isolation, prolonged activation, viral transduction, expansion culture, and quality testing. The overall process is lengthy, complex, and requires large viral inputs and stringent manufacturing conditions. Existing static processes still have significant limitations in applications requiring rapid infusion administration, point-of-care preparation, or reduced manufacturing costs.

[0003] Traditional static plate-well or culture bag transduction systems primarily rely on diffusion and gravity sedimentation. Under low multiples of infection (MOI) conditions, the probability of effective contact between the virus and target cells is significantly reduced, resulting in low viral transduction efficiency. To improve transduction efficiency, existing processes often employ methods such as increasing the viral input, extending co-incubation time, adding transduction-promoting reagents, or using centrifugation / dynamic culture. While these methods can improve viral transduction efficiency to some extent, they inevitably increase preparation costs and complicate the process flow, making closed-loop and automated integration more difficult, and also increasing the burden on subsequent processing. Currently, publicly available microfluidic transduction technologies still rely on static culture modes, shortening the diffusion distance between the virus and cells through spatial confinement and using high MOI to ensure viral infection efficiency. In these technologies, contact between the virus and T cells depends solely on passive diffusion and gravity sedimentation. Under low MOI conditions, the probability of effective contact remains limited, resulting in a significant decrease in transduction efficiency. Furthermore, the preparation cycle is long, making it difficult to meet the clinical needs of rapid drug delivery or point-of-care preparation. Summary of the Invention

[0004] The purpose of this invention is to provide a method, system, and application for rapid preparation of CAR-T cells using closed-loop microfluidics for low MOI lentiviral transduction, thereby overcoming the shortcomings of existing technologies. This invention involves short-term activation of T cells followed by mixing with a low MOI viral vector to form a cell-virus suspension. This suspension is then introduced into a closed-loop microfluidic system, where it is continuously circulated within a confined transduction channel. This significantly improves viral transduction efficiency under short-term T cell activation and low MOI conditions, thereby greatly reducing the preparation time of CAR-T cells.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a closed-loop microfluidic system for rapid preparation of CAR-T cells. The closed-loop microfluidic system includes: a reservoir, an inlet pipeline, a circulation drive unit, a bubble buffer / capture unit, a microfluidic chip, a return pipeline, and a temperature-controlled culture module. The reservoir is provided with a reservoir chamber, a sample loading / sampling port, a return port, and an outlet. The outlet is connected to the inlet of the circulation drive unit via the inlet pipeline, and the outlet of the circulation drive unit is connected to the inlet of the microfluidic chip via the bubble buffer / capture unit. The outlet of the microfluidic chip is connected to the return port of the reservoir via the return pipeline, forming a closed loop. The sampling / replacement interface is provided at the sample loading port of the reservoir and / or at a three-way valve, Luer connector, puncture port, or diaphragm interface on the inlet or return pipeline to realize the introduction of cell-virus suspension, sample extraction, culture medium replenishment, waste liquid discharge, and pipeline flushing.

[0006] Furthermore, the microfluidic chip has a shallow wide-channel confined transduction channel, which is selected from one or more shapes of serpentine channel, spiral channel, folded channel, wavy channel, and branching confluence channel.

[0007] Furthermore, the microfluidic chip has at least one inlet, at least one outlet, and at least one confined transduction channel.

[0008] The reservoir is used to temporarily store cell-virus suspensions and provide a buffer for circulating volume. The connecting tubing includes at least an inlet tubing and a return tubing, which are used to reconnect the reservoir, the circulation drive unit, the bubble buffer / capture unit, the microfluidic chip, and the reservoir into a closed loop, respectively. The circulation drive unit is used to provide continuous or intermittent circulation power. The sampling / liquid exchange interface is used for sample loading, sampling, replenishment, drainage, rinsing, or aseptic connection. The bubble buffer / capture unit is used to trap, aggregate, or release bubbles during circulation. The temperature-controlled culture module is used to maintain the temperature and gas environment required for T cell activation, virus transduction, and circulating culture.

[0009] The temperature-controlled culture module can be a 37°C, 5% CO2 incubator, a temperature-controlled base, a heating plate, a circulating water jacket, or a combination thereof, so that the liquid reservoir and the microfluidic chip are simultaneously in a constant temperature environment.

[0010] Secondly, the present invention provides a method for rapidly preparing CAR-T cells, the method comprising using the closed-loop microfluidic system as described in claim 1, the specific steps of which are as follows: T cells are mixed with a viral vector carrying the CAR gene to form a cell-virus suspension. The cell-virus suspension is introduced into the reservoir of a closed-loop microfluidic system through a sampling / replacement interface. The cell-virus suspension is continuously circulated within a closed loop formed by the microfluidic chip, the reservoir, and the connecting tubing by a circulation drive unit, repeatedly passing through a shallow, wide-channel confined transduction channel. After transduction, the cell-virus suspension in the closed loop is recovered through the sampling / replacement interface or the return port. The chip and connecting tubing are rinsed with culture medium to improve cell recovery rate and remove free viruses from the cell-virus suspension. The suspension is then transferred to a culture container for re-culture to obtain CAR-T cells.

[0011] This invention improves the effective contact frequency, cumulative contact time and unit virus utilization efficiency between the virus and T cells under low virus input conditions by continuously refluxing the cell-virus suspension system in a closed microfluidic loop, thereby obtaining CAR-T cell products with high transduction efficiency, good cell state and suitable for subsequent amplification and functional application.

[0012] Thirdly, the present invention provides the application of CAR-T cells prepared by the method described in the second aspect in the preparation of antitumor drugs.

[0013] This invention utilizes CAR-T cells obtained through the aforementioned closed-loop microfluidic rapid preparation method for the preparation of anti-tumor drugs. Because the CAR-T cells prepared by this method exhibit high CAR positivity, good cell viability, and in vitro killing function even under low MOI conditions, and because the preparation cycle is short and the viral load is low, the resulting drugs not only have reliable anti-tumor effects but also significantly reduce production costs and shorten patient waiting times. This makes them particularly suitable for rapid infusion, bedside preparation, and cost-sensitive clinical applications.

[0014] The core principle of this invention is the synergistic coupling of "microscale spatial confinement" and "closed-loop continuous reflux." During static culture, contact between virus particles and T cells mainly relies on Brownian motion, random diffusion, and finite sedimentation, resulting in low space utilization. Especially at low MOIs, a large number of viruses struggle to form effective contact with target cells within a limited time. This invention, by introducing the cell-virus system into a microscale confined channel, places the two within a shorter average separation distance and a more controlled local flow field, thereby physically increasing the probability of encounter per unit volume.

[0015] Furthermore, the closed-loop reflux mechanism allows viral particles and T cells that have not yet completed adsorption, fusion, endocytosis, or early transduction events to re-enter the transduction zone, forming multiple rounds of cumulative contact. Compared to unidirectional single-pass flow, the closed-loop system does not rely on a single instantaneous contact, but continuously increases the cumulative exposure dose and effective binding opportunity through repeated cycles. Compared to simple dynamic shaker or stirred culture, closed-loop microfluidics also provides a more stable channel scale, a shorter diffusion path, and more controllable flow rate / shear parameters, making it more suitable for achieving efficient transduction at low MOI.

[0016] Short-term activation complements this physical process at the biological state level. After 2-8 hours, preferably about 4 hours, of CD3 / CD28 stimulation, T cells can enter an early activation state suitable for viral entry and transgene expression, with cell membrane receptor expression, endocytic activity, and metabolic activity within a favorable range. However, if the activation time is too long, it may lead to increased cell stress, deeper differentiation, impaired short-term amplification, and a decline in subsequent functional state. Therefore, this invention does not simply combine activation and microfluidics side by side, but rather utilizes short-term activation to optimize cell transmissibility, and then utilizes closed-loop confined reflux to optimize virus-cell mass transfer and contact processes, thereby achieving synergistic technical effects under low MOI conditions.

[0017] Furthermore, by controlling the flow rate, cycle time, channel size, sample loading volume, cell density, viral input, and temperature control conditions, traditional open-loop viral transduction operations with significant batch variations can be transformed into a more standardized closed process, reducing the risk of contamination and facilitating expansion towards automation, miniaturization, bedside operation, and parallel scale-up.

[0018] Furthermore, the circulation drive unit can be a peristaltic pump, a syringe pump reciprocating drive module, a pressure drive module, a pneumatic pulse drive module, or a device with the same function; preferably, the circulation flow rate is 5-50 μL / min, more preferably 10-30 μL / min, and most preferably about 20 μL / min. 5-50 μL / min is an feasible range; below 10 μL / min, the number of cycles and cumulative contact frequency through the confined transduction channel per unit time are insufficient; above 30 μL / min, pumping pulsation, local shearing, bubble disturbance, and mechanical contact between cells and the tubing / chip wall increase, which may reduce cell viability, recovery rate, and short-term expansion capacity. Therefore, 10-30 μL / min is not the only range for obtaining CAR-T cells, but rather a preferred range that can stably obtain high transduction efficiency, high viability, and high unit virus utilization efficiency.

[0019] Furthermore, under the same T cell source, the same short-term activation conditions, the same MOI, and the same transduction duration, the closed-loop circulation flow rate was optimized. When the flow rate is too low, the cell-virus suspension circulates insufficiently through the confined transduction channel, reducing the cumulative effective contact opportunity between the virus and T cells. When the flow rate is too high, although the circulation frequency increases, local shearing and pumping disturbances increase, which may lead to a decrease in cell recovery rate, viability, and subsequent amplification capacity. Therefore, this invention uses 5-50 μL / min as an feasible range, 10-30 μL / min as a preferred range, and approximately 20 μL / min as a more preferred implementation condition, to balance transduction efficiency, cell viability, recovery rate, and virus output per unit input.

[0020] Furthermore, the method includes a rapid recovery culture step after transduction: the recovered cells are centrifuged or washed by membrane filtration to remove unbound viruses and impurities, and then transferred to a conventional culture container or amplification bag for continued culture; CAR positivity rate, cell viability, recovery rate, amplification fold, memory phenotype, cytokine secretion, and in vitro killing activity can be detected on Day 3, Day 5, or Day 7 to evaluate the quality and function of the cell product obtained by the method of the present invention.

[0021] Furthermore, the method is applicable to various immune cell engineering scenarios. Target cells can be peripheral blood-derived T cells or purified CD3+ cells. + T cells, γδ T cells, NK cells, or other immune cells that can accept the introduction of exogenous genes; the aim is to construct expression vectors for CAR, TCR, ChTCR, dual-target receptors, switch receptors, or other immune receptors. Therefore, this invention is not limited to specific targets or single receptor forms, but is applicable to a wide range of cell engineering preparation processes that require rapid viral transduction under low viral input conditions.

[0022] The microfluidic chip can be fabricated using PDMS, COC, COP, PMMA, glass, or their composite materials. It can incorporate one or more shallow, wide-channel transduction channels, and can be adapted to different loading volumes and cell masses through single-channel extension or parallel connection of multiple channels. Preferably, the single-channel width is 0.5-1.5 mm, the channel height is 50-200 μm, and the channel length is 40-100 cm; more preferably, the single-channel width is approximately 1.5 mm, the channel height is approximately 100 μm, and the channel length is approximately 60 cm, corresponding to an effective single-channel volume of approximately 90 μL. The single-channel flow rate is 5-50 μL / min, more preferably approximately 20 μL / min. The chip can be combined with surface hydrophilic treatment, anti-adsorption coating, or bubble management structures to improve system stability and cell recovery efficiency. This invention is not limited to a specific CAR configuration, but is preferably applicable to CAR expression cassettes containing an Igκ signal peptide, an FMC63-derived scFv, a CD8αhinge / transmembrane, a 4-1BB co-stimulatory domain, and a CD3ζ intracellular domain. It can also be extended to other CAR, TCR, or immune cell engineering vectors. Its applications include the rapid preparation of the final CAR-T cell product, as well as the development of intermediate processes, parameter screening, construction of virus-saving transduction processes, and the establishment of automated closed manufacturing platforms.

[0023] The beneficial effects of this invention are: (1) This invention addresses the problem of low effective contact between T cells and viruses and low infection efficiency in traditional static culture due to gravity settling to the bottom and viruses suspending in the culture medium. This invention co-cultures T cells and lentivirus suspensions in a closed-loop microfluidic system. The cell-virus suspension continuously circulates within a closed loop formed by the microfluidic chip, reservoir, and connecting tubing, repeatedly passing through the confined transduction channel. Under low MOI virus input conditions, this increases the effective contact frequency, cumulative contact time, and unit virus utilization efficiency between the virus and T cells, thereby obtaining CAR-T cell products with high transduction efficiency, good cell condition, and suitability for subsequent expansion and functional applications.

[0024] (2) The above-mentioned microfluidic device optimizes the preparation process of CAR-T cells, which can effectively reduce the amount of lentivirus used while ensuring high infection efficiency, thereby reducing the production cost of CAR-T cell therapy and alleviating the economic burden on patients.

[0025] (3) Compared with non-closed-loop microfluidic confined system, the present invention forms a truly closed loop through liquid reservoir, circulation drive unit, bubble buffer / capture unit, microfluidic chip and return pipeline, so that the same batch of cell-virus suspension passes through the confined transduction channel multiple times, thereby obtaining higher CAR positivity rate, better cell recovery rate and higher unit virus input utilization efficiency under low MOI conditions.

[0026] (4) This invention clarifies the coordination relationship between circulation flow rate, channel size, transduction time, number of circulation cycles, cell density and MOI, avoiding the need to improve transduction efficiency by simply increasing the viral input or extending the transduction time. Thus, even when viral input is limited, CAR-T cells with better CAR positivity, cell viability, recovery rate and functional status can still be stably obtained.

[0027] (5) The present invention provides a bubble buffer / capture unit and a sampling / liquid exchange interface, which can reduce the flow interruption, blockage, local shear increase or cell damage caused by bubbles entering the microfluidic chip, and facilitate the completion of sample loading, sampling, liquid replenishment, liquid drainage, rinsing and recovery operations under closed conditions, thereby improving process stability and repeatability.

[0028] (6) The closed-loop reflux structure of the present invention can be amplified in single or multiple channels in parallel according to the target cell quantity, and is suitable for the rapid preparation of CD19 CAR, B7-H3 CAR, IL13Rα2 CAR, dual-target CAR, ChTCR or other immune receptor engineered cells, and has the potential to be expanded to automated, miniaturized and bedside manufacturing platforms. Attached Figure Description

[0029] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0030] Figure 1 This is a schematic diagram of the closed-loop microfluidic system in Embodiment 1 of the present invention; Among them: 1. Liquid reservoir, 1-1. Liquid storage chamber, 1-2. Sample loading / sampling port, 1-3. Liquid return port, 2. Liquid inlet pipeline, 3. Circulation drive unit, 4. Bubble buffer / capture unit, 5. Microfluidic chip, 5-1. Shallow wide channel confined transduction channel, 6. Liquid return pipeline; Figure 2 A schematic diagram of the overall principle of using a closed-loop microfluidic system to assist in the rapid preparation of CAR-T cells; Figure 3 A timeline comparison chart of the microfluidic rapid manufacturing process and the conventional CAR-T manufacturing process; Figure 4 The graph shows the results of lentivirus transduction efficiency under low MOI conditions in microfluidic circulation. Figure 5 This figure compares the effects of static low-MOI transduction group, unidirectional low-MOI microfluidic flow transduction group, closed-loop low-MOI microfluidic circulation transduction group, and static high-MOI transduction group in CAR-T cell preparation; among them... Figure 5 A is a comparison chart of CAR positivity rates. Figure 5 B is a graph comparing cell viability. Figure 5 C is a comparison chart of recovery rates. Figure 5 D is a comparison chart of amplification folds over 72 hours. Figure 5 E is a comparison chart of the number of positive cells produced per unit of viral input (relative value). Figure 5 F is a comparison chart of in vitro killing activity (%, E:T = 5:1, 24 h); Figure 6This is a comparison diagram of the activation window and cell state; where, Figure 6 A is a comparison chart of inactive transducers. Figure 6 B is a comparison diagram of short-term activation transduction. Figure 6 C is a comparison of CAR positivity rates at different activation times. Figure 6 D is a comparison chart of survival rates. Figure 6 E is a comparison graph under 72 h amplification conditions. Figure 6 F is a conceptual diagram of a 24-hour microfluidic workflow; Figure 7 This is a graph showing the results of in vitro antitumor function verification; among them, Figure 7 A represents the Raji-Luc target cell lysis rate curves at different E:T ratios. Figure 7 B shows the results of IFN-γ cytokine secretion detection. Figure 7 C represents the results of IL-2 cytokine secretion detection. Figure 7 D is the result of TNF-α cytokine secretion detection; Figure 8 This is a schematic diagram of the chip's physical structure and confined transduction channels. Detailed Implementation

[0031] Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention. Specific conditions not specified in the embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Components whose manufacturers are not specified are all commercially available conventional products.

[0032] This invention provides a method, system, and application for rapid preparation of CAR-T cells using closed-loop microfluidics for low MOI lentiviral transduction. The method involves first briefly activating T cells, then introducing the virus into the T cells under low MOI conditions to form a cell-virus suspension. The suspension is then continuously refluxed and transduced using a closed-loop microfluidic system. This increases the effective contact frequency, cumulative contact time, and unit virus utilization efficiency between the virus and T cells under lower viral input conditions, thereby obtaining CAR-T cell products with high transduction efficiency, good cell condition, and suitability for subsequent expansion and functional applications.

[0033] The present invention adopts the following technical solution: This invention provides a closed-loop microfluidic system for rapid preparation of CAR-T cells. The closed-loop microfluidic system includes: a reservoir, an inlet line, a circulation drive unit, a bubble buffer / capture unit, a microfluidic chip, a return line, and a temperature-controlled culture module. The reservoir has a reservoir chamber, a sample loading / sampling port, a return port, and an outlet. The outlet is connected to the inlet of the circulation drive unit via the inlet line, the outlet of the circulation drive unit is connected to the inlet of the microfluidic chip via the bubble buffer / capture unit, and the outlet of the microfluidic chip is connected to the return port of the reservoir via the return line, forming a closed loop. The sampling / liquid exchange interface is located at the sample inlet of the reservoir and / or at a three-way valve, Luer connector, puncture port, or diaphragm interface on the inlet or return pipeline. That is, the sampling / liquid exchange interface can be a three-way valve, a four-way valve, a Luer connector, a self-sealing diaphragm port, or a puncture connector.

[0034] Furthermore, the liquid reservoir is a 1.5-15 mL sterile centrifuge tube, screw-cap bottle, syringe-type liquid reservoir, or soft bag-type liquid reservoir, preferably 1.5-5 mL.

[0035] Furthermore, the connecting tubing is medical silicone tubing, PharMed tubing, polytetrafluoroethylene tubing, or other biocompatible tubing, with an inner diameter of 0.3-1.6 mm, preferably 0.5-1.0 mm.

[0036] In one or more specific embodiments, the microfluidic chip has at least one shallow wide-channel confined transduction channel, which is selected from one or more combinations of shapes such as serpentine channel, spiral channel, folded channel, wavy channel, and branching confluence channel.

[0037] In one or more specific embodiments, the shallow wide-channel confined transduction channel has a width of 0.5-1.5 mm, a height of 50-200 μm, and a total length of 40-100 cm. Further, the shallow wide-channel confined transduction channel has a width of 1.5 mm, a height of 100 μm, and a total length of 60 cm. This shallow wide channel maintains microscale confinement in the height direction, keeping T cells and viral particles within a shorter vertical diffusion distance; simultaneously, compared to excessively narrow channels, a channel width of 0.5-1.5 mm reduces the risk of cell blockage and retention, improving cell suspension throughput and recovery rate. Taking a width of 1.5 mm, a height of 100 μm, and a length of 60 cm as an example, the effective volume of a single channel is approximately 90 μL, suitable for closed-loop continuous reflux transduction with a working volume of 0.2-2.0 mL.

[0038] Furthermore, the shallow wide channel does not rely on a single static confined culture, but rather serves as a repeatedly passing transduction zone under closed-loop circulation conditions; the reservoir and connecting tubing are used to hold working fluid larger than the effective volume of the channel, and the circulation drive unit allows the cell-virus suspension to enter the channel in batches, continuously, and repeatedly. Therefore, the effective volume of the channel is not equivalent to the total working volume of the system.

[0039] In one or more specific embodiments, the cyclic drive unit is selected from a peristaltic pump, a syringe pump reciprocating drive module, a pressure drive module, or a pneumatic pulse drive module.

[0040] In one or more specific embodiments, the bubble buffer / capture unit is one or a combination of a vertical expansion chamber, a hydrophobic and breathable membrane chamber, and a top exhaust port.

[0041] In one or more specific embodiments, the temperature-controlled culture module is an incubator, a temperature-controlled base, a heating plate, or a circulating water jacket; further, the temperature-controlled culture module is a 37°C, 5% CO2 incubator.

[0042] This invention also provides a method for rapidly preparing CAR-T cells, the method comprising using the aforementioned closed-loop microfluidic system, the specific steps of which are as follows: T cells are mixed with a viral vector carrying the CAR gene to form a cell-virus suspension. This suspension is then introduced into the reservoir of a closed-loop microfluidic system via a sampling / replacement interface. A circulation drive unit drives the cell-virus suspension to continuously circulate within a closed loop formed by the microfluidic chip, reservoir, and connecting tubing, repeatedly passing through a shallow, wide-channel confined transduction channel. After transduction, the cell-virus suspension is recovered from the closed loop via the sampling / replacement interface or return port to remove free viruses. The suspension is then transferred to a culture container for re-culturing, yielding CAR-T cells. The culture container can be a culture plate, culture flask, culture bag, or other conventional culture container. Further, if necessary, magnetic bead removal, cytokine supplementation, short-term amplification, or quality testing can be performed.

[0043] In one or more embodiments, the T cell pretreatment method is a short-term activation pretreatment, which includes short-term activation of T cells using CD3 / CD28 magnetic beads, coated antibodies, or artificial antigen-presenting particles, or functionally equivalent stimulation. The short-term activation time is 2-8 hours; further, the short-term activation time is 3-6 hours; even further, the short-term activation time is 4 hours. When CD3 / CD28 magnetic beads are used for short-term activation pretreatment, the bead-to-cell ratio is 0.5:1-3:1; further, the bead-to-cell ratio is 1:1.

[0044] In other embodiments, one or more of IL-2, IL-7, and IL-15 may be added to the culture system to keep T cells in an activated state that is conducive to viral entry and early expression, while avoiding excessive differentiation and decreased viability caused by prolonged activation.

[0045] In one or more embodiments, the T cells include, but are not limited to, undifferentiated T cells or engineered T cells; the T cells express CD3, CD4, or CD8, further expressing CD3; and even further, obtaining purified CD3. + T cells were resuspended at appropriate concentrations in serum-free or low-serum culture systems for later use. This involved collecting peripheral blood, apheresis leukocytes, or other samples containing T cells, and obtaining PBMCs or CD3-positive T cells through density gradient centrifugation, erythrocyte lysis, magnetic bead sorting, flow cytometry, or combinations thereof.

[0046] The viral vector includes, but is not limited to, lentiviruses or retroviruses, preferably lentiviruses.

[0047] In one or more embodiments, the MOI of the viral vector is 0.25-2.0; the T cell density is 0.5 × 10⁻⁶. 6 -20×10 6 The viral vector has an MOI of 0.5-1.0 and a T cell density of 1×10⁻⁶ cells / mL. 6 -10×10 6 per mL.

[0048] In one or more embodiments, the CAR-encoding gene includes, but is not limited to, encoding CD19 CAR, B7-H3 CAR, IL13Rα2 CAR, dual-target CAR, or ChTCR.

[0049] In one or more embodiments, the duration of a single transduction is 0.5-6 h, preferably 1-3 h; the total number of circulations is ≥10, preferably ≥30; and the circulation flow rate is 5-50 μL / min, preferably 10-30 μL / min, more preferably 20 μL / min.

[0050] In one or more embodiments, the invention may be carried out without relying on exogenous transduction agents, and pharmaceutically acceptable transduction additives may be selected, but these are not necessary conditions for the existence of the invention.

[0051] The present invention will be further described in detail below with reference to specific embodiments.

[0052] Example 1 A closed-loop microfluidic system, the schematic diagram of which is shown below. Figure 1 As shown. The system includes a liquid reservoir 1, a liquid inlet line 2, a circulation drive unit 3, a bubble buffer / capture unit 4, a microfluidic chip 5, a liquid return line 6, and a temperature-controlled culture module.

[0053] The reservoir 1 is provided with a reservoir chamber 1-1, a sample loading / taking port 1-2, a return port 1-3, and an outlet. The outlet is connected to the inlet of the circulation drive unit 3 via the inlet pipe 2, and the outlet of the circulation drive unit 3 is connected to the inlet of the microfluidic chip 5 via the bubble buffer / capture unit 4. The outlet of the microfluidic chip 5 returns to the reservoir's return port 1-3 via the return pipe 6.

[0054] The sampling / liquid exchange interface can be located at the sample loading / sampling port 1-2 of the reservoir, or at a three-way valve, four-way valve, Luer connector, self-sealing diaphragm port, or puncture connector on the inlet line 2 or return line 6, for sample loading, replenishment, sampling, cleaning, or venting. The bubble buffer / capture unit 4 is located downstream of the circulation drive unit 3 and upstream of the microfluidic chip 5. It can be a vertical expansion chamber, a hydrophobic and breathable membrane chamber, a top exhaust port, or a combination thereof, to eliminate pumping pulsation and trap bubbles, preventing bubbles from entering the shallow, wide-channel confined transduction channel 5-1 and causing flow interruption, cavitation, local shear increase, or cell damage.

[0055] The reservoir, bubble buffer / capture unit, and microfluidic chip are placed in a 37°C temperature-controlled environment. For systems using reservoirs or culture components with gas exchange capabilities, they are placed in a 37°C, 5% CO2 incubator to maintain stable culture medium temperature and pH. For closed, low-gas-exchange circulation loops, pre-equilibrated culture medium can be used, and transduction can be performed under 37°C temperature control. The circulation drive unit is located outside the incubator and connected to the internal components via sterile, sealed tubing.

[0056] In this embodiment, the microfluidic chip is a polydimethylsiloxane (PDMS)-glass composite chip, and a serpentine (or foldback) shallow wide channel is provided inside the chip. Figure 1 The diagram illustrates a single-channel structure. The shallow, wide-channel confined transduction channel 5-1 has a width of 1.5 mm, a height of 100 μm, and a total length of 60 cm. Based on the example parameters, the effective volume of the single channel is approximately 90 μL.

[0057] Figure 1 The direction of the middle arrow indicates the direction of the circulation flow of the cell-virus suspension, that is, it enters the circulation drive unit 3 through the inlet pipe 2 from the reservoir 1, then enters the microfluidic chip 5 through the bubble buffer / capture unit 4, flows through the shallow wide channel confined transduction channel 5-1 in the chip, and returns to the reservoir 1 through the return pipe 6, thus forming a closed circulation loop.

[0058] The closed-loop microfluidic system operates as follows: Short-term activated T cells are mixed with low-MOI lentivirus and injected into reservoir 1. Powered by the circulation drive unit 3, the cell-virus suspension flows through inlet line 2 and bubble buffer / capture unit 4 into the shallow, wide-channel confined transduction channel 5-1 of the microfluidic chip 5. After flowing through the channel, it returns to reservoir 1 via return line 6, forming a closed loop. During circulation, the suspension repeatedly passes through the shallow, wide-channel confined transduction channel 5-1, allowing the virus and T cells to enter the microscale transduction region multiple times under controlled flow conditions, thereby shortening the effective diffusion distance and increasing cumulative contact opportunities. Simultaneously, the bubble buffer / capture unit 4 reduces the risk of flow interruption, increased local shear, or cell damage caused by bubbles entering the chip. The temperature-controlled culture module maintains the required temperature and culture environment for transduction. After reaching the preset circulation time or number of cycles, the cells are recovered and recultured to obtain CAR-T cells.

[0059] like Figure 3 As shown, the timeline difference between the rapid CAR-T cell manufacturing process using a closed-loop microfluidic system and the conventional CAR-T manufacturing process is illustrated. The conventional CAR-T manufacturing process typically includes T cell isolation, activation for approximately 24-48 hours or longer, static viral transduction, multi-day amplification culture, and subsequent quality control, with key manufacturing steps being time-consuming. In contrast, this embodiment employs a 4-hour short-term activation combined with closed-loop microfluidic cyclic transduction, increasing the cumulative frequency of virus-T cell contact and the unit virus utilization efficiency under low MOI conditions, thereby compressing the key activation and transduction steps to approximately 20-24 hours.

[0060] exist Figure 3In the corresponding experimental design, the MF-rmCAR-T group used 4-hour short-term activation, low-MOI lentiviral loading, and closed-loop microfluidic transduction; the cmCAR-T group used conventional activation and static transduction procedures. The two groups were compared in terms of CAR positivity, cell viability, amplification capacity, memory phenotype, and in vitro function under the same T cell source, the same CAR vector, and the same detection time points. Figure 3 It can be seen that the MF-rmCAR-T group showed a significant reduction in time, and this embodiment can still maintain effective transduction and cell function while shortening the preparation time of CAR-T cells.

[0061] Example 2 The method for rapidly preparing CAR-T cells based on the closed-loop microfluidic system in Example 1, such as Figure 2 As shown, it includes the following steps: Step S1: Sample Acquisition and Cell Isolation Peripheral blood was collected from donors, and peripheral blood mononuclear cells (PBMCs) were separated using density gradient centrifugation. CD3+ cells were then obtained using a CD3-positive magnetic bead selection method. + T cells. Isolated CD3 + T cells at 1×10 6 The cells / mL were resuspended in TexMACS medium and supplemented with IL-2 (100 U / mL) for later use.

[0062] Step S2: Short-time activation pretreatment The CD3 prepared in step S1 + T cells were mixed with CD3 / CD28 magnetic beads at a 1:1 bead-to-cell ratio and activated at 37°C and 5% CO2 for 4 h.

[0063] Step S3: Establishment of Low MOI Virus Loading and Suspension System Concentrated lentivirus carrying CD19 CAR was used as a vector, with MOI set to 1. T cells activated for 4 h were thoroughly mixed with concentrated lentivirus carrying CD19 CAR to form a cell-virus suspension with a total volume of 1.0 mL.

[0064] Step S4: Closed-loop microfluidic continuous reflux transduction The cell-virus suspension obtained in step S3 is introduced into reservoir 1 through sampling / replacement interfaces 1-3. The circulation drive unit 3 is then activated, causing the suspension to continuously circulate within a closed loop and repeatedly pass through the shallow, wide-channel confined transduction channel 5-1 within the chip. The single-channel flow rate is 20 μL / min, and the circulation is maintained at 37°C for 180 min. Under these conditions, the total throughput is approximately 3.6 mL, and the channel volume is updated approximately 40 times based on the effective channel volume.

[0065] Step S5: Cell Recovery and Resuscitation. After transduction, the cell suspension was recovered through sampling / medium exchange interfaces 1-3, and the chip and tubing were rinsed with fresh culture medium to improve cell recovery rate. Free lentivirus was then removed by centrifugation at 300×g for 5 min, and the cells were resuspended in IL-2-containing medium for resuscitation to obtain the final CAR-T cell product.

[0066] Example 3 The difference compared to Example 2 is that the single-channel flow rate is set to 10 μL / min.

[0067] Example 4 The difference compared to Example 2 is that the single-channel flow rate is set to 30 μL / min.

[0068] Example 5 The difference compared to Example 2 is that the MOI is set to 0.5.

[0069] Comparative Example 1 The difference compared to Example 2 is that the short-time activation time is set to 0 h.

[0070] Comparative Example 2 The difference compared to Example 2 is that the short-time activation time is set to 19 h.

[0071] Comparative Example 3 The difference compared to Example 2 is that the single-channel flow rate is set to 5 μL / min.

[0072] Comparative Example 4 The difference compared to Example 2 is that the single-channel flow rate is set to 50 μL / min.

[0073] Comparative Example 5 Methods for preparing CAR-T cells via static low MOI transduction: Separate CD3 according to steps S1 and S2 of Example 2. + T cells were activated for a short period of 4 hours. Using the same CD19 CAR lentiviral vector and low MOI conditions (MOI=1) as in Example 2, cells and virus were mixed to form a cell-virus suspension with a total volume of 1.0 mL. This suspension was added to the wells of a standard culture plate or culture vessel and statically incubated at 37°C for 180 min; no microfluidic channels or reflux were performed during this process. After transduction, static low MOI control CAR-T cells were obtained using the same washing, recovery culture, and detection procedures as in Example 2.

[0074] Comparative Example 6 A method for preparing CAR-T cells using unidirectional microfluidic low MOI: Following steps S1 to S3 of Example 2, a low-MOI cell-virus suspension, activated for 4 hours (MOI=1), with a total volume of 1.0 mL, was obtained. The suspension was then delivered at a flow rate of 20 μL / min through a shallow, wide-channel confined transduction channel with the same structure and dimensions as in Example 1, and collected from the chip outlet into a separate sterile container. The collected solution was not returned to the reservoir, thus not forming a closed-loop circulation. After unidirectional passage, the collected solution was maintained at 37°C until the total transduction treatment time reached 180 min, ensuring the total treatment time was consistent with Example 2. Unidirectional microfluidic low-MOI control CAR-T cells were obtained using the same washing, recovery culture, and detection procedures as in Example 2.

[0075] Comparative Example 7 Methods for preparing CAR-T cells via static high MOI transduction: CD3+ T cells were isolated and activated for 4 hours following steps S1 and S2 of Example 2. Using the same CD19 CAR lentiviral vector as in Example 2, the viral input was increased to a high MOI condition, forming a cell-virus suspension with a total volume of 1.0 mL; the high MOI condition should be based on the viral input amount used in the actual experimental records. The suspension was added to the wells of a conventional culture plate or culture container and statically incubated at 37°C for 180 min, without passing through a microfluidic channel or undergoing reflux. Static high MOI control CAR-T cells were obtained using the same washing, recovery culture, and detection procedures as in Example 2.

[0076] The CAR-T cells prepared in Examples 2-5 and Comparative Examples 1-7 were tested for indicators including CAR positivity rate, cell viability, cell recovery rate, 72-hour amplification fold, the number of positive cells produced per unit of virus input, and in vitro killing activity.

[0077] The testing conditions and procedures for the indicator detection are as follows: (1) Grouping and variable control: Examples 2-5 and Comparative Examples 1-7 all used CD3+ T cells from the same source, the same CAR vector, the same culture medium, and the same recovery culture conditions. Except for the activation time, circulation flow rate, flow mode, or virus input conditions that were clearly changed in each example or comparative example, all other conditions remained the same.

[0078] (2) CAR positivity rate and cell viability: After transduction and recovery culture for 72 h, cells were collected and the proportion of CAR-positive cells was detected by flow cytometry; cell viability was detected simultaneously using live / dead dyes. The CAR positivity rate was expressed as the proportion of CAR-positive cells within the viable cell phylum, and the cell viability was expressed as the proportion of viable cells to the total number of cells detected.

[0079] (3) Cell recovery rate and 72-h amplification fold: The cell recovery rate was detected after transduction and after the chip and tubing were rinsed, and was calculated as "number of recovered live cells / number of live cells introduced at the beginning of transduction × 100%"; the 72-h amplification fold was calculated as "number of live cells at 72 hours of recovery culture / number of live cells recovered at the end of transduction".

[0080] (4) Positive cell output per unit of virus input: calculated as “CAR positive live cell count / virus input” and normalized to 1.0 using the detection value of static low MOI transduction group (comparative example 5) to evaluate the positive cell output efficiency under unit virus input conditions.

[0081] (5) In vitro killing activity: The recovered CAR-T cells and Raji-Luc target cells were co-cultured for 24 h at E:T=5:1. The target cell lysis rate was calculated by luciferase signaling or equivalent cell activity detection method, and the results were expressed as a percentage.

[0082] (6) Replication setup and result recording: Each detection group should be independently replicated at least three times or validated using three independent donor cells, and the original experimental records, flow cytometry files and statistical results should be used as the basis for the data in Table 1 and the attached figures.

[0083] The results of CAR-T cell marker detection are shown in Table 1.

[0084] Table 1. Detection results of different CAR-T cell markers

[0085] Test experiments and results: like Figure 4 The results show the experimental results of improving lentiviral transduction efficiency under low MOI conditions using microfluidic circulation. Primary human T cells were selected and activated briefly for 4 hours with CD3 / CD28 magnetic beads. Static plate groups and closed-loop microfluidic chip groups were then set up. Both groups used the same T cell source, cell concentration, lentiviral vector, MOI, and infection time. MOIs were set to 0.5 and 1.0 to examine whether the closed-loop microfluidic system in Example 1 could improve transduction efficiency under limited viral input conditions. After transduction, the cells were cultured back to the preset detection time point, and CAR positivity or GFP positivity was detected by flow cytometry. Figure 4 Results A showed that in the E19 system, the CAR positivity rate of the chipset at MOI 0.5 was approximately 13.8% ± 2.8%, higher than that of the plate group (5.1% ± 2.1%); the CAR positivity rate of the chipset at MOI 1.0 was approximately 21.8% ± 1.5%, higher than that of the plate group (13.9% ± 2.0%). Figure 4As shown in Figure B, the chipset was also observed to outperform the static group under low MOI conditions in the ECS1 standalone virus system. These results indicate that, under low viral input conditions, closed-loop microfluidic circulation can improve lentiviral transduction efficiency by increasing the repeated co-localization and cumulative contact opportunities between the virus and T cells.

[0086] like Figure 5 As shown, four groups of process comparison experiments were conducted. Group A was the static low MOI transduction group, where the cell-virus suspension was statically co-incubated in a conventional well plate or culture container; Group B was the unidirectional low MOI microfluidic flow transduction group, where the cell-virus suspension passed through the microfluidic channel once or a limited number of times without forming a closed-loop reflux; Group C was the closed-loop low MOI microfluidic circulation transduction group, where the closed-loop system consisting of the reservoir, inlet tubing, circulation drive unit, bubble buffer / capture unit, microfluidic chip, and return tubing described in Example 1 was repeatedly circulated; Group D was the static high MOI transduction group, used to evaluate the effect of simply increasing the viral input on transduction efficiency and cell quality. Groups A, B, and C used low MOI conditions, while Group D used higher MOI conditions; except for the flow mode and MOI setting, all other conditions were kept consistent across the four groups, including T cell source, short-term CD3 / CD28 activation method, culture medium system, transduction time, recovery culture conditions, and detection time points. Detection indicators included CAR positivity rate (…). Figure 5 A) Cell viability ( Figure 5 B), Cell recovery rate ( Figure 5 C), 72 h amplification fold ( Figure 5 D) Number of positive cells produced per unit of viral input ( Figure 5 E) and in vitro killing activity ( Figure 5 F). Figure 5 The results showed that the CAR positivity rate in the closed-loop low MOI group C was approximately 34.0%, which was higher than that in group A (15.0%) and group B (24.0%), and close to that in group D (36.0%) with static high MOI. The cell viability in group C was approximately 91.0%, the recovery rate was approximately 88.0%, and the fold increase after 72 h was approximately 2.9, all of which were better than or close to those of other groups. Figure 5 E shows that group C had the highest output of positive cells per unit of viral input (relative value of 2.6). Figure 5 The F-test showed that the in vitro killing activity of group C (68%) was close to that of the static high MOI group (70.0%), indicating that closed-loop continuous reflux can maintain high transduction efficiency and functional activity while reducing viral input.

[0087] The influence of circulation flow rate parameters was further investigated. Under the same T cell source, same 4-hour short-term activation, same MOI=1.0, and same circulation time of 180 min, only the closed-loop circulation flow rate was changed, setting five groups: 5 μL / min, 10 μL / min, 20 μL / min, 30 μL / min, and 50 μL / min. The results showed that the 5 μL / min group had a lower CAR positivity rate and lower viral output per unit flow rate due to insufficient circulation through the confined transduction pathway per unit time. The 10-30 μL / min range could better balance CAR positivity rate, cell viability, and recovery rate, with 20 μL / min being the optimal condition. Although CAR-T cells could still be obtained in the 50 μL / min group, cell viability, recovery rate, and 72-hour expansion capacity decreased. This indicates that flow rates below 10 μL / min or above 30 μL / min do not necessarily prevent the acquisition of CAR-T cells, but it is difficult to consistently achieve the high transduction efficiency, high cell viability, high recovery rate, and good functional state achieved by the preferred technical solution of this application.

[0088] like Figure 6 As shown, the activation window and cell state were compared in an experiment. Three groups were set up: an inactive group, a short-term activated group, and a long-term activated group. The inactive group underwent viral transduction without CD3 / CD28 stimulation; the short-term activated group received CD3 / CD28 magnetic bead stimulation for 4 hours; and the long-term activated group received conventional or extended activation for more than 19 hours. The three groups were compared in CAR positivity rate, cell viability, and 72-hour amplification capacity under the same cell source, MOI, microarray structure, infection time, and detection time. Figure 6 A and Figure 6 B is used to compare the difference in chip transduction efficiency under inactive and short-term activated conditions; Figure 6 C was used to compare the CAR positivity rate at different activation times; Figure 6 D is used to compare cell viability; Figure 6 E was used to compare amplification capacity at 72 h. Figure 6 F was used to demonstrate the 24-hour microfluidic workflow concept. Results showed that the CAR positivity rate of unactivated T cells remained low after infection at MOI=1 for 45, 90, 135, or 180 min. After short-term activation (around 4 h), the chip-based group showed significantly higher positivity than the static plate group at different infection durations. While long-term activation could enhance some activation-related states, it led to a decrease in cell viability and short-term expansion capacity. Therefore, short-term activation of approximately 4 h achieves a better balance between transduction efficiency, cell viability, and expansion potential.

[0089] like Figure 7As shown, the in vitro killing function verification experiment was conducted. MF-rmCAR-T, cmCAR-T, and untransduced T cells were co-cultured with Raji-Luc target cells at a predetermined E:T ratio, preferably E:T = 0.5:1, 1:1, 2.5:1, or 5:1. Tumor cell lysis rate and cytokine secretion levels were detected after 24 h or 72 h. Tumor lysis rate was calculated using luciferase signal transduction, flow cytometry, or other cell viability assays; IFN-γ, IL-2, and TNF-α were measured using ELISA, CBA, or other cytokine detection methods. Figure 7 As shown in Figure A, at an E:T ratio of 0.5:1, the tumor lysis rate mediated by MF-rmCAR-T was approximately 65.5% ± 10.7%, higher than that of cmCAR-T (46.5% ± 10.9%); at an E:T ratio of 1:1, the lysis rate of MF-rmCAR-T was approximately 88.3% ± 8.5%, higher than that of cmCAR-T (62.5% ± 11.1%). Cytokine detection results ( Figure 7 B~7D) also showed that the rapid microfluidic-controlled group was superior to or no less effective than the conventionally manufactured group in terms of IFN-γ, IL-2, and TNF-α secretion. These results demonstrate that the closed-loop microfluidic rapid preparation process of this invention can not only improve the transduction efficiency of low MOI cells, but also obtain CAR-T cell products with effective anti-tumor functions.

[0090] like Figure 8 As shown, the chip's physical structure and shallow, wide-channel confined transduction channel configuration are illustrated. The chip can be fabricated using PDMS bonded to glass, or using COC, COP, PMMA, glass, or their composite materials. The chip internally features serpentine, zigzag, wavy, spiral, or branching confluence shallow, wide channels, preferably serpentine or zigzag channels. The channel width is 0.5-1.5 mm, preferably 1.0-1.5 mm, more preferably about 1.5 mm; the channel height is 50-200 μm, more preferably about 100 μm; and the total channel length is 40-100 cm, more preferably about 60 cm. Taking a width of 1.5 mm, a height of 100 μm, and a length of 60 cm as an example, the effective volume of a single channel is approximately 90 μL. The chip has at least one inlet and at least one outlet. The inlet is connected to the outlet of the bubble buffer / capture unit, and the outlet returns to the reservoir via a return line. Figure 8The display shows the relative positions of the chip body, inlet, outlet, and shallow wide-channel confined transduction channel. This channel, with a height of 50-200 μm, restricts the vertical diffusion distance of the cell-virus suspension; simultaneously, its 0.5-1.5 mm width reduces cell blockage, retention, and recovery difficulties. Under closed-loop circulation conditions, viruses and T cells that have not yet completed the adsorption or entry process can repeatedly enter the transduction region, thereby increasing the effective contact frequency. The chip can be configured with single or multiple parallel channels depending on the target cell volume, and can be combined with anti-adsorption coatings, surface hydrophilic treatments, or bubble management structures to improve system stability and cell recovery efficiency.

[0091] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A closed-loop microfluidic system for rapid preparation of CAR-T cells, characterized in that, The closed-loop microfluidic system includes: a liquid reservoir, an inlet pipeline, a circulation drive unit, a bubble buffer / capture unit, a microfluidic chip, a return pipeline, and a temperature-controlled culture module; The liquid reservoir is provided with a liquid storage chamber, a sample loading / taking port, a return port, and a liquid outlet; the liquid outlet is connected to the inlet of the circulation drive unit via an inlet pipe, the outlet of the circulation drive unit is connected to the inlet of the microfluidic chip via a bubble buffer / capture unit, and the liquid outlet of the microfluidic chip is connected to the return port of the liquid reservoir via a return pipe, thus forming a closed circulation loop; The sampling / liquid exchange interface is located at the sample inlet of the liquid reservoir and / or at a three-way valve, Luer connector, puncture port, or diaphragm interface on the inlet or return pipeline.

2. The closed-loop microfluidic system according to claim 1, characterized in that, The microfluidic chip has a shallow wide-channel confined transduction channel, which is selected from one or more shapes of serpentine channel, spiral channel, foldback channel, wavy channel, and branch-and-converge channel. Preferably, the shallow wide-channel confined transduction channel has a width of 0.5-1.5 mm, a height of 50-200 μm, and a total length of 40-100 cm; More preferably, the shallow wide-channel confined transduction channel has a width of 1.5 mm, a height of 100 μm, and a total length of 60 cm.

3. The closed-loop microfluidic system according to claim 1, characterized in that, The cycle drive unit is selected from one of the following: a peristaltic pump, a syringe pump reciprocating drive module, a pressure drive module, or a pneumatic pulse drive module. Alternatively, the bubble buffer / capture unit may be one or a combination of a vertical expansion chamber, a hydrophobic and breathable membrane chamber, and a top exhaust port.

4. The closed-loop microfluidic system according to claim 1, characterized in that, The temperature-controlled culture module is an incubator, a temperature-controlled base, a heating plate, or a circulating water jacket; Preferably, the temperature-controlled culture module is a 37°C, 5% CO2 incubator.

5. A method for rapidly preparing CAR-T cells, characterized in that, The method includes using the closed-loop microfluidic system of claim 1, and the specific steps are as follows: T cells are mixed with a viral vector carrying the CAR gene to form a cell-virus suspension. The cell-virus suspension is introduced into the reservoir of a closed-loop microfluidic system through a sampling / replacement interface. The cell-virus suspension is continuously circulated within a closed loop formed by the microfluidic chip, the reservoir, and the connecting tubing by a circulation drive unit, repeatedly passing through a shallow, wide-channel confined transduction channel. After transduction, the cell-virus suspension in the closed loop is recovered through the sampling / replacement interface or the return port to remove free viruses. The suspension is then transferred to a culture container for re-culturing to obtain CAR-T cells.

6. The method according to claim 5, characterized in that, The T cells were pretreated using a short-term activation pretreatment method, which included stimulation with CD3 / CD28 magnetic beads, coated antibodies, or artificial antigen-presenting particles; the short-term activation time was 2-8 hours. Preferably, the short-time activation time is 3-6 hours; more preferably, the short-time activation time is 4 hours.

7. The method according to claim 5, characterized in that, The T cells include undifferentiated T cells or engineered T cells; the T cells express CD3, CD4 or CD8, preferably CD3; The viral vector includes lentivirus or retrovirus, preferably lentivirus.

8. The method according to claim 5, characterized in that, The viral vector had an MOI of 0.25-2.0; the T cell density was 0.5 × 10⁻⁶. 6 -20×10 6 cells / mL; Preferably, the MOI of the viral vector is 0.5-1.0; the T cell density is 1×10⁻⁶. 6 -10×10 6 cells / mL; Preferably, the CAR-encoding gene includes a CD19 CAR, B7-H3 CAR, IL13Rα2 CAR, dual-target CAR, or ChTCR.

9. The method according to claim 5, characterized in that, The duration of a single transduction is 0.5-6 h, preferably 1-3 h; The total number of cycles is ≥10, preferably ≥30; The circulation flow rate is 5-50 μL / min, preferably 10-30 μL / min, and more preferably 20 μL / min.

10. The use of CAR-T cells prepared by the method according to any one of claims 5-9 in the preparation of antitumor drugs.

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