A method for rapid isolation of high activity immune cells
By preparing microdroplets using microfluidic technology and utilizing Matrigel to inhibit cell migration, rapid separation of highly active immune cells was achieved. This solves the problem of the inability to effectively separate highly active and inactive immune cells in existing technologies, and improves cell activity and separation efficiency.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- ACCURATE INT BIOTECHNOLOGY (GUANGZHOU) CO LTD
- Filing Date
- 2026-01-27
- Publication Date
- 2026-05-29
AI Technical Summary
Existing methods for isolating immune cells cannot effectively separate highly active and inactive immune cells, leading to apoptosis of inactive cells and the release of apoptosis factors. Furthermore, these methods are cumbersome, time-consuming, and result in cells with low viability.
Microfluidic technology was used to prepare microdroplets containing immune cells. By preparing microdroplets in a matrix gel suspension, the gelled Matrigel was used to inhibit cell migration, so that the migration rate of highly active immune cells was faster than that of low-activity immune cells, thus achieving rapid separation of highly active immune cells.
It enables rapid separation of highly active immune cells with a cell viability rate of over 95%, and features fast separation speed and simple operation.
Smart Images
Figure CN121574918B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of cell biology and immunology, and in particular to a method for rapidly isolating highly active immune cells. Background Technology
[0002] Immune cell isolation technology is an important tool in biological and medical research, widely used for the isolation and purification of cells in biological samples. Existing techniques in this field typically utilize methods such as magnetic bead separation and flow cytometry to isolate immune cells. However, these methods only separate the immune cells themselves, failing to separate highly active from less active immune cells. Less active immune cells are highly susceptible to apoptosis, releasing apoptosis factors, which in turn lead to the apoptosis of highly active immune cells. Furthermore, methods such as magnetic bead separation and flow cytometry are cumbersome, time-consuming, and yield immune cells with low viability. Therefore, there is an urgent need for a method that achieves faster separation speed and yields cells with higher viability. Summary of the Invention
[0003] To address the technical problems existing in the prior art, the present invention aims to provide a method for rapidly separating highly active immune cells. This method utilizes microfluidic technology to rapidly separate highly active immune cells.
[0004] According to a first aspect of the present invention, the present invention provides a method for rapidly isolating highly active immune cells, comprising the following steps:
[0005] (1) Connect the microfluidic chip to the microfluidic device, add the cell suspension containing immune cells to the microfluidic chip, and prepare cell-containing microdroplets through the microfluidic device;
[0006] (2) Add the cell-containing microdroplets from step (1) to the culture medium to obtain a mixture, shake it to obtain a microdroplet suspension, culture it, and collect the cells that crawl out of the microdroplets during the culture process to obtain highly active immune cells.
[0007] In some implementations, a cell suspension containing immune cells is added into the cell pores of the microfluidic chip.
[0008] In some implementations, the oil pores of the microfluidic chip are pre-injected with fluorinated oil (Flou-Oil) before the cell suspension is added.
[0009] In some implementations, the cell-containing microdroplets in step (1) are collected into a collection tube via the microdroplet outlet of the chip.
[0010] In some implementations, the microfluidic chip in step (1) is a microfluidic microdroplet chip; the diameter of the microdroplet is 90-250 μm.
[0011] In some embodiments, the diameter of the microdroplets containing mixed cells in step (1) ranges from 90-130 μm, 90-150 μm, 90-160 μm, 100-170 μm, 120-180 μm or 130-200 μm.
[0012] In some embodiments, in step (1), after connecting the microfluidic chip to the microfluidic device, fluorinated oil containing surfactant is added to the oil hole of the microfluidic chip, the microfluidic device is started, the working pressure is set to 150-400 Pa, and then a cell suspension containing immune cells is added to the oil hole to obtain cell-containing microdroplets; the mass percentage of surfactant in the fluorinated oil containing surfactant is 1-5 wt%.
[0013] In some embodiments, the preparation of the cell suspension containing immune cells in step (1) includes:
[0014] Cell clusters containing immune cells are added to a mixture of culture medium and Matrigel, mixed well, and a cell suspension is obtained.
[0015] In some implementations, the manufacturer of the microfluidic chip in step (1) is Suzhou Hanguang Micro-Nano Technology Co., Ltd., and the model is Droplet Production Chip 80-130 D.
[0016] The chip has an oil inlet (Oil pore), cell pores, and a microdroplet outlet; a fluorinated oil Flou-Oil containing 2% wt surfactant is used as the oil phase and stored in an oil storage chamber, and the oil pore and the oil phase are connected by a hose (Tygon hose); the Tygon hose has a specification of 0.02*0.06*0.02 inches.
[0017] In some implementations, Matrigel is a matrix gel manufactured by Corning Incorporated, Yisheng Biotechnology Co., Ltd., MCE and Gino Biotechnology, etc.
[0018] In some embodiments, the immune cells in step (1) are tumor-infiltrating lymphocytes; the volume of the culture medium is 10%-30% of the Matrigel volume.
[0019] In some embodiments, the cell density in the cell suspension containing immune cells in step (1) ranges from 10-1. 5 Up to 10 8 The number of immune cells per ml was 10. 5 Up to 10 7 per ml.
[0020] In some embodiments, the cell cluster containing immune cells contains only immune cells. If it contains only immune cells, the method provided by the present invention can separate highly active immune cells from less active immune cells, thereby obtaining highly active immune cells.
[0021] In some embodiments, the culture medium is a tumor-infiltrating lymphocyte culture medium; the preparation of the tumor-infiltrating lymphocyte culture medium includes:
[0022] Anti-CD3 antibody, anti-CD28 antibody, and IL-2 were added to X-VIVO 15 medium and mixed well to obtain the tumor-infiltrating lymphocyte culture medium; the volume ratio of anti-CD3 antibody to X-VIVO 15 medium was 1:600-1200; the volume ratio of anti-CD28 antibody to X-VIVO 15 medium was 1:600-1200; and the volume ratio of IL-2 to X-VIVO 15 medium was 1:600-1500.
[0023] In some embodiments, the volume ratio of the cell suspension containing immune cells in step (1) to the culture medium in step (2) is 5:80-500;
[0024] In some implementations, the shaking process in step (2) is carried out in an environment with a temperature of 36.5-37.5°C and containing 4.5-5.5% CO2;
[0025] In some embodiments, the shaking process in step (2) includes: adding fluorinated oil containing surfactant to the mixture, then shaking for 15-40 minutes at a speed of 60-70 rpm, then shaking for 2-4 hours at a speed of 70-120 rpm, and letting it stand for 3-5 minutes to separate the fluorinated oil from the microdroplets to obtain a microdroplet suspension; the volume ratio of the fluorinated oil containing surfactant to the mixture is 1-10:400.
[0026] In some embodiments, the surfactant in the surfactant-containing fluorinated oil is 2 wt% by mass.
[0027] In some implementations, the shaking process described in step (2) is performed in a sterile incubator.
[0028] In some embodiments, the temperature of the culture treatment in step (2) is 36.5-37.5°C and the culture treatment time is 24-96h.
[0029] In some embodiments, in step (2), the cells that have crawled out of the microdroplets during the culture process are collected by using a filter with a pore size of 30-100 μm to collect the cells that have crawled out of the microdroplets during the 24-96 hours of culture.
[0030] In some embodiments, cells that have crawled out of microdroplets during the 24-96 hour culture process are collected using a filter with a pore size of 30-100 μm. The filtrate and the filter material on the filter are collected separately to obtain highly active immune cells (filtrate) and microdroplets (filter material) respectively. Alternatively, the microdroplets can be separated from the highly active immune cells by natural sedimentation.
[0031] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0032] The method provided by this invention uses a microfluidic device to prepare microdroplets containing immune cells in a matrix gel suspension. The gelled Matrigel inhibits cell migration, allowing highly active immune cells to migrate faster than less active ones, thus achieving rapid separation of highly active immune cells. The immune cells separated using this method exhibit high activity, with a viability rate exceeding 95%. Furthermore, this method is fast and simple to operate. Attached Figure Description
[0033] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0034] Figure 1 The fluorescence result of the immune cell-containing fluid obtained in step (2) of Comparative Example 1 in the test of Effect Verification 1 is shown.
[0035] Figure 2 The fluorescence image of the microdroplet suspension obtained after separating fluorinated oil in step (11) of Example 1 is shown in the test of Effect Verification 1.
[0036] Figure 3 The fluorescence image of the microdroplet suspension obtained after separating fluorinated oil in step (11) of Example 2 is shown in the test of Effect Verification 1.
[0037] Figure 4 The fluorescence image of the microdroplet suspension obtained after separating fluorinated oil in step (11) of Example 3 is shown in the test of Effect Verification 1.
[0038] Figure 5 The result of flow spectroscopy detection in effect verification 2 is shown for the microdroplet suspension obtained after separating fluorinated oil in step (11) of Example 1. Detailed Implementation
[0039] To better understand the technical solution of the present invention, the embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the described embodiments are merely some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0040] The main reagents and consumables used in the following examples are shown in Table 1 below.
[0041] Table 1
[0042]
[0043] The culture medium used in the following examples is a tumor-infiltrating lymphocyte culture medium, and the preparation method includes:
[0044] Anti-CD3 antibody, anti-CD28 antibody, and IL-2 were added to X-VIVO 15 medium and mixed thoroughly to obtain tumor-infiltrating lymphocyte culture medium; the volume ratio of anti-CD3 antibody to X-VIVO 15 medium was 1:1000; the volume ratio of anti-CD28 antibody to X-VIVO 15 medium was 1:1000; and the volume ratio of IL-2 to X-VIVO 15 medium was 1:1200.
[0045] Comparative Example 1
[0046] A method for sorting immune cells using CD45 magnetic beads includes the following steps:
[0047] (1) Immune cells purchased from Cellenion were reselected using 10 ml of culture medium to obtain a TIL cell suspension with a cell density of 2 × 10⁻⁶ cells / mL. 6 To obtain TIL cell suspension, 5 ml of TIL cell suspension was transferred to a 15 ml centrifuge tube and centrifuged for 3 min at 1500 rpm. The supernatant was discarded, and the suspension was resuspended in 300 μl of tumor-infiltrating lymphocyte culture medium. 5 μl of CD45 antibody was added, and the mixture was incubated at 37°C for 10 min. After rehydration, 5 μl of CD45 magnetic beads were added, and the mixture was incubated at 37°C for 10 min. After rehydration, the mixture was placed on a magnetic rack at room temperature for 3-5 min, and the supernatant was discarded. 500 μl of Release Buffer was added, and the mixture was rehydrated and incubated at room temperature for 3-5 min. After rehydration, the mixture was placed on a magnetic rack at room temperature for 3-5 min, and the supernatant was transferred to a 15 ml centrifuge tube. 3.5 ml of culture medium was added for resuspending, and the mixture was centrifuged and the supernatant was discarded. 5 ml of PBS buffer was added for washing, and the mixture was centrifuged 3 times. The suspension was then resuspended in 4 ml of culture medium, transferred to a low-adsorption 6-well plate, and then transferred to an incubator for culture.
[0048] (2) At intervals of 24h, 48h and 72h, the cells at the bottom were collected to obtain liquid containing immune cells and their activity was tested.
[0049] The activity of immune cells was detected using acridine orange (AO) / propidium iodide (PI) dyes. The viability of immune cells obtained in this example was 80% (the detection method was based on the article "Methodological Validation of Killer Cell Count and Viability Detection Based on AO / PI Fluorescent Staining Principle and Study on Cell Culture, Cryopreservation and Resuscitation Stability").
[0050] Example 1
[0051] A rapid isolation protocol for highly active immune cells includes the following steps:
[0052] (1) Weigh out 3M Novec HFE7500 fluorinated oil (TS-3M-HFE7500-500), put it into a 15ml centrifuge tube, and store it in the dark for later use;
[0053] (2) Open the software program of the microfluidic device, connect the power supply, and turn on the switches of the microfluidic vacuum pump, air compressor and pressure controller, set the pressure to 0 mBar, and prepare;
[0054] (3) Take one microfluidic chip with a specification of 80-130μm (microfluidic chips with a specification of 80-130μm can be used to prepare microdroplets with a diameter of 80-130μm, the same below), one 2ml collection tube, and then connect the oil storage chamber and the chip oil inlet with a microfluidic Tygon tubing. At the same time, connect the microdroplet outlet and the collection tube with a microfluidic Tygon tubing. Add 100μl of fluorinated oil Flou-Oil to the collection tube, and then connect the collection tube to the pressure controller and check its airtightness. In addition, use a constant temperature plate to maintain the chip temperature at 4℃.
[0055] (4) Add anti-CD3 antibody, anti-CD28 antibody and IL-2 to X-VIVO 15 medium, mix well to obtain tumor-infiltrating lymphocyte culture medium; the volume ratio of anti-CD3 antibody to X-VIVO 15 medium is 1:1000; the volume ratio of anti-CD28 antibody to X-VIVO 15 medium is 1:1000; the volume ratio of IL-2 to X-VIVO 15 medium is 1:800.
[0056] (5) The immune cells purchased from Cellenion were resuspended in 10 ml of culture medium to obtain a TIL cell suspension with a cell density of 2 × 10⁻⁶ cells / mL. 6To obtain cells per ml, aspirate 5 ml of TIL cell suspension and place it in a 15 ml centrifuge tube. Centrifuge for 3 min at 1500 rpm, discard the supernatant, resuspend in 1 ml of culture medium, add 1 μl of cellTracker™ green CMFDA fluorescent dye, incubate at 37°C for 30 min, centrifuge and discard the supernatant, wash with 5 ml of PBS buffer, centrifuge 3 times, resuspend in 5 ml of PBS buffer, mix well, and count the cells in the suspension.
[0057] (6) Take 9 million cells after counting in step (1), then centrifuge at 1500 rpm for 3 min, discard the supernatant, resuspend in 30 μl of tumor infiltrating lymphocyte culture medium, and transfer to 1.5 ml sterile PE tube. Then add 170 μl of Corning™ Matrigel, mix well to obtain cell / Matrigel suspension, and store at 4°C for later use.
[0058] (7) Take 20 ml of tumor-infiltrating lymphocyte culture medium and add it to a 100 mm cell culture dish. Let it stand at room temperature for later use.
[0059] (8) Take 2 ml of fluorinated oil Flou-Oil and put it into the oil storage chamber. Take 100 μl of fluorinated oil Flou-Oil and add it into the cell well of the chip. Then, adjust the pressure to 150 mBar. After purging the air from the chip and the tubing channel, adjust the pressure to 0 mBar.
[0060] (9) Add the cell / Matrigel suspension from step (6) into the cell well of the chip, let it stand for 2-3 minutes, then adjust the pressure to 300 mBar for 10 minutes. After the cell / Matrigel suspension in the cell well of the chip is drained, adjust the pressure to 0 mBar.
[0061] (10) Take out the collection tube and transfer it to a sterile laminar flow hood. Then transfer the microdroplets in the collection tube to the culture dish in step (7), add 100 μl of fluorinated oil (Flou-Oil) to spread the microdroplets out completely. Then transfer the culture dish to a shaker in a 37°C incubator and shake it at 60 rpm. After 40 min, adjust the speed to 90 rpm and continue shaking.
[0062] (11) Shake for 3 hours until all microdroplets settle to the bottom. Then transfer the culture dish to a sterile laminar flow hood and transfer the microdroplet suspension at the bottom of the dish to a 50ml centrifuge tube. After mixing, let it stand for 5 minutes until the fluorinated oil Flou-Oil separates from the microdroplets. Then transfer the upper microdroplet suspension to a new 50ml centrifuge tube and shake it 10 times to obtain the microdroplet suspension after separating the fluorinated oil. Transfer the microdroplet suspension after separating the fluorinated oil to a low-adsorption 6-well plate and then to an incubator for incubation.
[0063] Cells that have crawled out of the microdroplets at intervals of 24h, 48h, and 72h were collected (filtered using a 70μm pore size filter, and the filtrate was collected) to obtain highly active immune cells.
[0064] The activity of immune cells was detected using acridine orange (AO) / propidium iodide (PI) dyes. The viability of immune cells obtained in this example was 98% (the detection method was based on the method described in the article "Methodological Validation of Killer Cell Count and Viability Detection Based on AO / PI Fluorescent Staining Principle and Study on Cell Culture, Cryopreservation and Resuscitation Stability").
[0065] Example 2
[0066] A rapid isolation protocol for highly active immune cells includes the following steps:
[0067] (1) Weigh out 3M Novec HFE7500 fluorinated oil (TS-3M-HFE7500-500), put it into a 15ml centrifuge tube, and store it in the dark for later use;
[0068] (2) Open the software program of the microfluidic device, connect the power supply, and turn on the switches of the microfluidic vacuum pump, air compressor and pressure controller, set the pressure to 0 mBar, and prepare;
[0069] (3) Take a microfluidic chip with a specification of 80-130μm and a 2ml collection tube. Then connect the oil storage chamber and the chip oil inlet with a microfluidic Tygon tubing. At the same time, connect the microdroplet outlet and the collection tube with a microfluidic Tygon tubing. Add 100μl of fluorinated oil Flou-Oil to the collection tube. Then connect the collection tube to the pressure controller and check its airtightness. In addition, use a constant temperature plate to maintain the chip temperature at 4℃.
[0070] (4) Add anti-CD3 antibody, anti-CD28 antibody and IL-2 to X-VIVO 15 medium, mix well to obtain tumor-infiltrating lymphocyte culture medium; the volume ratio of anti-CD3 antibody to X-VIVO 15 medium is 1:1000; the volume ratio of anti-CD28 antibody to X-VIVO 15 medium is 1:1000; the volume ratio of IL-2 to X-VIVO 15 medium is 1:800.
[0071] (5) The immune cells purchased from Cellenion were resuspended in 10 ml of culture medium to obtain a TIL cell suspension with a cell density of 2 × 10⁻⁶ cells / mL. 6 To obtain cells per ml, aspirate 5 ml of TIL cell suspension and place it in a 15 ml centrifuge tube. Centrifuge for 3 min at 1500 rpm, discard the supernatant, resuspend in 1 ml of culture medium, add 1 μl of cellTracker™ green CMFDA fluorescent dye, incubate at 37°C for 30 min, centrifuge and discard the supernatant, wash with 5 ml of PBS buffer, centrifuge 3 times, resuspend in 5 ml of PBS buffer, mix well, and count the cells in the suspension.
[0072] (6) Take 6 million cells after counting in step (1), then centrifuge at 1500 rpm for 3 min, discard the supernatant, resuspend in 30 μl of tumor infiltrating lymphocyte culture medium, and transfer to a 1.5 ml sterile PE tube. Then add 170 μl of Corning™ Matrigel, mix well to obtain cell / Matrigel suspension, and store at 4°C for later use.
[0073] (7) Take 20 ml of tumor-infiltrating lymphocyte culture medium and add it to a 100 mm cell culture dish. Let it stand at room temperature for later use.
[0074] (8) Take 2 ml of fluorinated oil Flou-Oil and put it into the oil storage chamber. Take 100 μl of fluorinated oil Flou-Oil and add it into the cell well of the chip. Then, adjust the pressure to 150 mBar. After purging the air from the chip and the tubing channel, adjust the pressure to 0 mBar.
[0075] (9) Add the cell / Matrigel suspension from step (6) into the cell well of the chip, let it stand for 3 min, then adjust the pressure to 280 mBar for 20 min. After the cell / Matrigel suspension in the cell well of the chip is drained, adjust the pressure to 0 mBar.
[0076] (10) Take out the collection tube and transfer it to a sterile laminar flow hood. Then transfer the microdroplets in the collection tube to the culture dish in step (7), add 100 μl of fluorinated oil (Flou-Oil) to spread the microdroplets out completely. Then transfer the culture dish to a shaker in a 37°C incubator and shake it at 60 rpm. After 40 min, adjust the speed to 90 rpm and continue shaking.
[0077] (11) Shake for 3 hours until all microdroplets settle to the bottom. Then transfer the culture dish to a sterile laminar flow hood and transfer the microdroplet suspension at the bottom of the dish to a 50ml centrifuge tube. Gently blow to mix and let stand for 5 minutes until the fluorinated oil Flou-Oil separates from the microdroplets. Then transfer the upper microdroplet suspension to a new 50ml centrifuge tube and shake 10 times to obtain the microdroplet suspension after separating the fluorinated oil. Transfer the microdroplet suspension after separating the fluorinated oil to a low-adsorption 6-well plate and then to an incubator for incubation.
[0078] Cells that have crawled out of the microdroplets at intervals of 24h, 48h, and 72h were collected (using a 70μm pore size filter and taking the filtrate) to obtain highly active immune cells.
[0079] The activity of immune cells was detected using acridine orange (AO) / propidium iodide (PI) dyes. The viability of immune cells obtained in this example was 95% (the detection was performed according to the method described in the article "Methodological Validation of Killer Cell Count and Viability Detection Based on AO / PI Fluorescent Staining Principle and Study on Cell Culture, Cryopreservation and Resuscitation Stability").
[0080] Example 3
[0081] A rapid isolation protocol for highly active immune cells includes the following steps:
[0082] (1) Weigh out 3M Novec HFE7500 fluorinated oil (TS-3M-HFE7500-500), put it into a 15ml centrifuge tube, and store it in the dark for later use;
[0083] (2) Open the software program of the microfluidic device, connect the power supply, and turn on the vacuum pump, air compressor and pressure controller of the microfluidic device. Set the pressure to 0 mBar to prepare.
[0084] (3) Take one chip with a specification of 80-130μm and one 2ml collection tube. Then connect the oil storage chamber and the chip oil inlet with a microfluidic Tygon tubing. At the same time, connect the microdroplet outlet and the collection tube with a microfluidic Tygon tubing. Add 100μl of fluorinated oil Flou-Oil to the collection tube. Then connect the collection tube to the pressure controller and check its airtightness. In addition, use a constant temperature plate to maintain the chip temperature at 4℃.
[0085] (4) Add anti-CD3 antibody, anti-CD28 antibody and IL-2 to X-VIVO 15 medium, mix well to obtain tumor-infiltrating lymphocyte culture medium; the volume ratio of anti-CD3 antibody to X-VIVO 15 medium is 1:1000; the volume ratio of anti-CD28 antibody to X-VIVO 15 medium is 1:1000; the volume ratio of IL-2 to X-VIVO 15 medium is 1:800.
[0086] (5) The immune cells purchased from Cellenion were resuspended in 10 ml of culture medium to obtain a TIL cell suspension with a cell density of 2 × 10⁻⁶ cells / mL. 6 To obtain cells per ml, aspirate 5 ml of TIL cell suspension and place it in a 15 ml centrifuge tube. Centrifuge for 3 min at 1500 rpm, discard the supernatant, resuspend in 1 ml of culture medium, add 1 μl of cellTracker™ green CMFDA fluorescent dye, incubate at 37°C for 30 min, centrifuge and discard the supernatant, wash with 5 ml of PBS buffer, centrifuge 3 times, resuspend in 5 ml of PBS buffer, mix well, and count the cells in the suspension.
[0087] (6) Take 3 million cells after counting in step (1), then centrifuge at 1500 rpm for 3 min, discard the supernatant, resuspend in 30 μl of tumor infiltrating lymphocyte culture medium, and transfer to 1.5 ml sterile PE tube. Then add 170 μl of Corning™ Matrigel, mix well to obtain cell / Matrigel suspension, and store at 4°C for later use.
[0088] (7) Take 20 ml of tumor-infiltrating lymphocyte culture medium and add it to a 100 mm cell culture dish. Let it stand at room temperature for later use.
[0089] (8) Take 2 ml of fluorinated oil Flou-Oil and put it into the oil storage chamber. Take 100 μl of fluorinated oil Flou-Oil and add it into the cell well of the chip. Then, adjust the pressure to 150 mBar. After purging the air from the chip and the tubing channel, adjust the pressure to 0 mBar.
[0090] (9) Add the cell / Matrigel suspension from step (6) into the cell well of the chip, let it stand for 3 minutes, then adjust the pressure to 250 mBar for about 10-20 minutes. After the cell / Matrigel suspension in the cell well of the chip is drained, adjust the pressure to 0 mBar.
[0091] (10) Take out the collection tube and transfer it to a sterile laminar flow hood. Then transfer the microdroplets in the collection tube to the culture dish in step (7), add 100 μl of fluorinated oil (Flou-Oil) to spread the microdroplets out completely. Then transfer the culture dish to a shaker in a 37°C incubator and shake it at 60 rpm. After 40 min, adjust the speed to 90 rpm and continue shaking.
[0092] (11) Shake for 3 hours until all microdroplets settle to the bottom. Then transfer the culture dish to a sterile laminar flow hood and transfer the microdroplet suspension at the bottom of the dish to a 50ml centrifuge tube. Gently blow to mix and let stand for 5 minutes until the fluorinated oil Flou-Oil separates from the microdroplets. Then transfer the upper microdroplet suspension to a new 50ml centrifuge tube and shake 10 times to obtain the microdroplet suspension after separating the fluorinated oil. Transfer the microdroplet suspension after separating the fluorinated oil to a low-adsorption 6-well plate and then to an incubator for incubation.
[0093] Cells that have crawled out of the microdroplets at intervals of 24h, 48h, and 72h were collected (using a 70μm pore size filter and taking the filtrate) to obtain highly active immune cells.
[0094] The activity of immune cells was detected using acridine orange (AO) / propidium iodide (PI) dyes. The viability of immune cells obtained in this example was 93% (the detection was performed according to the method described in the article "Methodological Validation of Killer Cell Count and Viability Detection Based on AO / PI Fluorescent Staining Principle and Study on Cell Culture, Cryopreservation and Resuscitation Stability").
[0095] Effect Verification 1:
[0096] (1) Take 500 μl of the immune cell-containing liquid obtained in step (2) of Comparative Example 1 and transfer it to a new low-adsorption 6-well plate for culture (add 2 mL of tumor-infiltrating lymphocyte culture medium). At each time point of 0 h, 24 h, 48 h and 72 h, after shaking well, take 100 μl of the suspension into a low-adsorption 96-well plate, add 0.1 μl of acridine orange (AO) / propidium iodide (PI) dye, incubate at 37 °C for 30 min, and then use an EOS M7000 microscope for fluorescence tracking and activity detection (the detection was performed according to the method described in the article "Verification of the Methodology for Detection of Killer Cell Quantity and Viability Based on AO / PI Fluorescence Staining Principle and Study on the Stability of Cell Culture, Cryopreservation and Resuscitation"; the same applies below). The results are as follows. Figure 1 As shown, the viability of immune cells at 0h was 75%, and the viability of immune cells after 24h tended to be 80%.
[0097] (2) Take 500 μl of the microdroplet suspension obtained after separating fluorinated oil in step (11) of Example 1 and transfer it to a new low-adsorption 6-well plate for culture (add 2 mL of tumor-infiltrating lymphocyte culture medium). At each time point of 0 h, 24 h, 48 h and 72 h, after shaking well, take 100 μl of the suspension into a low-adsorption 96-well plate, add 0.1 μl of acridine orange (AO) / propidium iodide (PI) dye, incubate at 37 °C for 30 min, and then use an EOS M7000 microscope for fluorescence tracking and activity detection. The results are as follows. Figure 2 As shown, most immune cells were able to migrate out of the microdroplets, and the viability of the migrated immune cells was 98%. In addition, the mortality rate of immune cells that did not migrate out of the microdroplets was as high as 95%.
[0098] (3) Take 500 μl of the microdroplet suspension obtained after separating the fluorinated oil in step (11) of Example 2 and transfer it to a new low-adsorption 6-well plate for culture (add 2 mL of tumor-infiltrating lymphocyte culture medium). At each time point of 0 h, 24 h, 48 h and 72 h, after shaking well, take 100 μl of the suspension into a low-adsorption 96-well plate, add 0.1 μl of acridine orange (AO) / propidium iodide (PI) dye, incubate at 37 °C for 30 min, and then use an EOS M7000 microscope for fluorescence tracking and activity detection. The results are as follows. Figure 3 As shown, most immune cells were able to migrate out of the microdroplets, and the viability of the migrated immune cells was over 95%. In addition, the mortality rate of immune cells that did not migrate out of the microdroplets was as high as 96%.
[0099] (4) Take 500 μl of the microdroplet suspension obtained after separating fluorinated oil in step (11) of Example 3 and transfer it to a new low-adsorption 6-well plate for culture (add 2 mL of tumor-infiltrating lymphocyte culture medium). At each time point of 0 h, 24 h, 48 h and 72 h, after shaking well, take 100 μl of the suspension to a low-adsorption 96-well plate, add 0.1 μl of acridine orange (AO) / propidium iodide (PI) dye, incubate at 37 °C for 30 min, and then use an EOS M7000 microscope for fluorescence tracking and activity detection. The results are as follows. Figure 4 As shown, most immune cells were able to migrate out of the microdroplets, and the viability of the migrated immune cells was over 93%. In addition, the mortality rate of immune cells that did not migrate out of the microdroplets was as high as 95%.
[0100] Effect Verification 2:
[0101] The microdroplet suspension obtained after separating fluorinated oil in step (11) of Example 1 was incubated in a 5% CO2 incubator at 37°C for 72 hours. Then, 5 ml of sample solution was aspirated into a 15 ml centrifuge tube, filtered through a 70 μm sieve, and the filtrate was collected. The filtrate was centrifuged, the supernatant was removed, and the precipitate was collected.
[0102] Mix the precipitate from step (1) with 100 μl of flow cytometry washing solution, and add 1 μl of CD4 antibody and 1 μl of CD8 antibody. Incubate at room temperature in the dark for 20 min.
[0103] Centrifuge and wash twice with added flow cleaning solution.
[0104] Resuspend in 1 ml of flow cytometry washing buffer, and then analyze using a cell flow cytometer.
[0105] Cell flow cytometry results as follows Figure 5 As shown, CD4 cells accounted for approximately 81.5% and CD8 cells accounted for approximately 8.1%. This result indicates that the level of helper T cells is extremely high, accounting for 81.5%, while the level of suppressor T cells is low, at only 8.1%. The helper T cells are much higher than the suppressor T cells, indicating that the tumor-infiltrating lymphocytes isolated using the method of this invention have extremely strong immune activity. This also further proves that the immune cell isolation method of this invention can rapidly isolate highly active immune cells.
[0106] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.
Claims
1. A method for rapidly isolating highly active immune cells, characterized in that, Includes the following steps: (1) Connect the microfluidic chip to the microfluidic device, add the cell suspension containing immune cells to the microfluidic chip, and prepare microdroplets through the microfluidic device; (2) Add the microdroplets described in step (1) to the culture medium to obtain a mixture, shake it to obtain a microdroplet suspension, culture it, collect the cells that crawl out of the microdroplets during the culture process, and obtain highly active immune cells; The diameter of the microdroplets in step (1) is 90-250 μm; In step (1), after connecting the microfluidic chip to the microfluidic device, fluorinated oil containing surfactant is added to the oil hole of the microfluidic chip, the microfluidic device is started, and the working pressure is set to 250 mBar, 280 mBar or 300 mBar. Then, the cell suspension is added to the oil hole to obtain microdroplets. The preparation of the cell suspension containing immune cells in step (1) includes: Cell clusters containing immune cells are added to a mixture of culture medium and Matrigel, mixed well, and a cell suspension is obtained. The immune cells mentioned in step (1) are tumor-infiltrating lymphocytes; the volume of the culture medium is 10%-30% of the Matrigel volume; In step (1), the total cell density in the cell suspension ranges from 10. 5 Up to 10 8 The number of immune cells per ml was 1.5 × 10⁻⁶. 7 cells / ml, 3.0×10 7 pcs / ml or 4.5×10 7 cells / mL; The shaking treatment in step (2) includes: adding fluorinated oil containing surfactant to the mixture, then shaking for 15-40 minutes at a speed of 60-70 rpm, then shaking for 2-4 hours at a speed of 70-120 rpm, and letting it stand for 3-5 minutes to separate the fluorinated oil from the microdroplets to obtain a microdroplet suspension; the volume ratio of the fluorinated oil containing surfactant to the mixture is 1-10:
400. The culture treatment time in step (2) is 24-96 hours.
2. The method for rapidly isolating highly active immune cells according to claim 1, characterized in that, The microfluidic chip mentioned in step (1) is a microfluidic microdroplet chip.
3. The method for rapidly isolating highly active immune cells according to claim 1, characterized in that, In the fluorinated oil containing surfactant, the mass percentage of surfactant is 1-5 wt%.
4. The method for rapidly isolating highly active immune cells according to claim 1, characterized in that, The culture medium is a tumor-infiltrating lymphocyte culture medium; the preparation of the tumor-infiltrating lymphocyte culture medium includes: Anti-CD3 antibody, anti-CD28 antibody, and IL-2 were added to X-VIVO 15 medium and mixed well to obtain the tumor-infiltrating lymphocyte culture medium; the volume ratio of anti-CD3 antibody to X-VIVO 15 medium was 1:600-1200; the volume ratio of anti-CD28 antibody to X-VIVO 15 medium was 1:600-1200; and the volume ratio of IL-2 to X-VIVO 15 medium was 1:600-1500.
5. The method for rapidly isolating highly active immune cells according to claim 1, characterized in that, The volume ratio of the cell suspension containing immune cells in step (1) to the culture medium in step (2) is 5:80-500; the shaking treatment in step (2) is carried out in an environment with a temperature of 36.5-37.5℃ and containing 4.5-5.5% CO2.
6. The method for rapidly isolating highly active immune cells according to claim 1, characterized in that, The temperature for the culture treatment in step (2) is 36.5-37.5℃.
7. The method for rapidly isolating highly active immune cells according to claim 1, characterized in that, In step (2), the cells that crawled out of the microdroplets during the culture process were collected by using a filter with a pore size of 30-100 μm to collect the cells that crawled out of the microdroplets during the 24-96 hours of culture.