Tumor infiltration lymphocyte sorting system based on negative pressure microfluidics

Abstract

The invention discloses a tumor infiltrating lymphocyte sorting system based on negative pressure microfluidics in the technical field of tumor infiltrating lymphocyte preparation, the tumor infiltrating lymphocyte sorting system comprises a microfluidic chip and a controller, and the microfluidic chip is provided with a sample injection layer, a pre-sorting layer, a capture layer, a dynamic detection layer and a culture layer which are communicated in sequence. Target TIL is enriched and captured through different antibody fixing technologies, mechanical damage to cells is reduced through negative pressure microfluidics, then the cells are sorted and eluted and enter different culture rooms to be directly cultured, integration of the process and automation of operation are improved, complexity and errors of manual operation are reduced, and therefore the requirements of clinical large-scale and standard application are met.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor infiltrating lymphocyte preparation, and in particular is a tumor infiltrating lymphocyte sorting system based on negative pressure microfluidics. Background Art

[0002] Adoptive cellular immunotherapy (ACT) shows great potential in cancer treatment. Its core goal is to restore and expand highly efficient tumor-infiltrating lymphocytes (TILs) to enhance the immune system's ability to attack tumors. However, TILs in tumors are currently scarce and difficult to isolate. This dilemma severely restricts the improvement of treatment efficacy and the optimization of therapeutic doses, significantly hindering the clinical application and promotion of ACT therapy.

[0003] Traditional TIL sorting technology mainly relies on fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS). FACS achieves cell sorting through droplet formation and high-speed fluid dynamics, but this technology has many disadvantages: its droplet formation process is unstable and the scanning accuracy is insufficient, resulting in a large number of cell losses; during the sorting process, the cell droplets need to be electrified, and the strong electric field environment can easily damage cell activity, making the in vitro expansion of the sorted TIL cells take a long time, about 10 weeks, accounting for 80% of the entire TIL therapy production cycle, ultimately leading to cell function exhaustion and significantly reducing its anti-tumor effect. Magnetic-activated cell sorting (MACS) uses magnetic labels and separation columns to achieve cell sorting, but a large number of dead cells and debris often remain in the separation columns, making it difficult to completely remove them, resulting in insufficient TIL purity and affecting subsequent treatment effects. In addition, both of these traditional technologies find it difficult to achieve a good balance between high throughput and high recovery rate, and cannot meet the needs of large-scale clinical applications.

[0004] Through the development of a combination of antibodies and magnetic beads, existing technologies have made it possible to more conveniently use microfluidic chips to sort TILs and even their subpopulations, as shown in the paper "Efficient recovery of potent tumor-infiltrating lymphocytes through quantitative immunomagnetic cell sorting." However, in addition to problems with the sorting technology itself, existing tumor-infiltrating lymphocyte sorting systems also have obvious defects in the overall process. The entire sorting process often needs to be completed in steps, including sorting, amplification, and functional verification. The processes between each link are scattered, which can easily introduce contamination or errors, greatly affecting the accuracy and reliability of the sorting results. At the same time, the existing system has a low degree of automation and is highly dependent on manual operation. This not only limits the realization of high-throughput sorting, but also increases the risk of human error, reduces work efficiency and stability, and is difficult to meet the requirements of large-scale, standardized clinical applications.

[0005] Therefore, it is necessary to propose a tumor-infiltrating lymphocyte sorting system based on negative pressure microfluidics that can accurately sort tumor-infiltrating lymphocytes, ensure cell survival rate and sorting purity, and complete sorting, amplification and functional verification on a microfluidic chip, thereby improving the efficiency of automated sorting, reducing errors and enhancing reproducibility. Summary of the Invention

[0006] In order to solve the above problems, the purpose of the present invention is to provide a tumor-infiltrating lymphocyte sorting system based on negative pressure microfluidics, which enriches and captures target TILs through different antibody fixation technologies, reduces mechanical damage to the cells through negative pressure microfluidics, and then sorts and elutes them into different culture chambers for direct culture, thereby improving the integration of the process and the automation of operations, reducing the tediousness and errors of manual operations, and thus meeting the needs of large-scale, standardized clinical applications.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows: a tumor-infiltrating lymphocyte sorting system based on negative pressure microfluidics, comprising a microfluidic chip and a controller, wherein the microfluidic chip is provided with a sample introduction layer, a pre-sorting layer, a capture layer, a dynamic detection layer, and a culture layer that are sequentially connected;

[0008] The injection layer is used to access the tumor cell suspension and sorting buffer and mix the two;

[0009] The pre-sorting layer is used to pre-sort TILs using CD3 antibodies;

[0010] The capture layer is used to sort pre-sorted tumor-infiltrating lymphocytes using a combination of CD8 and CD39 antibodies;

[0011] The dynamic detection layer is used to detect TIL exhaustion status and TIL metabolic activity using fluorescence detection technology and impedance flow detection technology;

[0012] The culture layer is used to directly access pre-sorted TILs for direct culture.

[0013] The principle of the basic scheme is: the present invention accesses the tumor cell suspension and sorting buffer through the sampling layer and mixes them to reduce mechanical damage; the pre-sorting layer uses CD3 antibodies to perform preliminary sorting of TILs; the capture layer combines CD8 antibodies and CD39 antibodies to further sort the pre-sorted TILs; the dynamic detection layer uses fluorescence detection technology and impedance flow detection technology to detect the exhaustion state and metabolic activity of TILs; finally, the culture layer is directly connected to the pre-sorted TILs for culture. The entire process uses negative pressure microfluidics technology to accurately control the cell movement trajectory, avoid mechanical damage, and improve sorting efficiency and cell activity.

[0014] The basic solution offers the following benefits: 1. The combination of a multi-layer microfluidic chip structure and specific antibodies enables efficient sorting of tumor-infiltrating lymphocytes, improving sorting purity and recovery rate. Negative pressure microfluidics technology controls cell movement, avoiding mechanical and electrical damage common in traditional sorting techniques, while maintaining cell activity and function.

[0015] 3. The integration of sorting, detection, and culture functions on a single microfluidic chip reduces the tedious steps in traditional sorting processes, reduces contamination risks, and improves operational convenience and automation. The dynamic detection layer enables real-time analysis of TIL exhaustion and metabolic activity, helping to optimize sorting thresholds and assess cell function, providing important data support for subsequent cell expansion and treatment.

[0016] 4. Through rapid sorting and an optimized culture environment, the in vitro expansion time of TILs is shortened, reducing the risk of cell function exhaustion and helping to improve the effectiveness of adoptive cell immunotherapy. Compared with traditional sorting technologies, this system is expected to shorten the production cycle of TIL therapies, reduce treatment costs, and improve treatment accessibility.

[0017] Furthermore, the sample introduction layer includes an injection hole opened on the microfluidic chip, the injection hole is connected to the injection chamber, the injection chamber is connected to a "Y"-shaped tube, another branch of the "Y"-shaped tube is connected to the liquid injection chamber, the liquid injection chamber is connected to the injection hole, and the trunk of the "Y"-shaped tube is connected to a tortuous delay channel;

[0018] The pre-sorting layer includes a pre-sorting chamber, which is connected to the delay channel. Several microcolumns are fixedly connected to the inner wall of the pre-sorting chamber. The microcolumns are coated with a polydopamine coating, and the polydopamine coating is covalently fixed with CD3 antibodies. The other side of the pre-sorting chamber is connected to a pre-sorting tube. The middle of the pre-sorting tube is connected to a first reversing valve, and the first reversing valve is connected to a waste liquid hole.

[0019] The beneficial effects of the basic solution are as follows: the design of the sample injection layer achieves efficient mixing of the tumor cell suspension and the sorting buffer through a "Y"-shaped tube structure and a tortuous delay channel, reducing mechanical damage to the cells during the injection process and improving cell activity. The pre-sorting chamber, in which the microcolumns are coated with a polydopamine coating and immobilized with CD3 antibodies, can specifically capture TILs expressing the CD3 antigen, achieving preliminary cell enrichment. The setting of the first reversing valve facilitates the control of waste liquid discharge, reduces cross-contamination, and improves the purity and efficiency of sorting. This design not only optimizes the initial steps of cell sorting and ensures the accuracy of the subsequent sorting process, but also lays the foundation for the efficient operation of the entire sorting system by reducing cell damage and improving sorting purity.

[0020] Furthermore, the capture layer includes several capture tubes spirally wound around each other, each of which is connected to the pre-sorting tube. The capture tubes are connected to each other by several inclined connecting tubes, and the inclination direction of the connecting tubes is all toward the dynamic detection layer. The inner walls of the capture tubes are spirally fixed with capture strips made of nanofibers, and different capture antibodies are fixed on the capture strips, including CD8 antibodies and CD39 antibodies.

[0021] The beneficial effects of the basic solution are: the capture layer effectively extends the flow path of cells in the capture area through the spirally wound capture tube and inclined connecting tube design, increases the contact time between cells and capture antibodies, and improves capture efficiency. The capture strips made of nanofibers are fixed with CD8 and CD39 antibodies, which can specifically capture the pre-sorted target TIL subpopulations to achieve precise sorting, and the capture strips interfere with the originally stable laminar flow in the capture tube. Combined with the continuous diversion of the connecting tube and the spiral flow of the capture tube, the cell suspension is turned into a chaotic vortex, further increasing the probability of contact between cells and capture antibodies. The inclined connecting tube is facing the dynamic detection layer, which facilitates the exchange of cell suspensions in different capture tubes. Overall, the design of the capture layer significantly improves the sorting accuracy of TIL, while reducing the residue of dead cells and debris, improving the sorting purity, and providing high-quality cell populations for subsequent cell function testing and culture.

[0022] Furthermore, the dynamic detection layer includes a detection chamber, one side of the detection chamber is connected to the capture tube, the other side of the detection chamber is connected to the output tube, and the connection point between the detection chamber and the output tube is symmetrically fixed with an impedance electrode;

[0023] The other end of the output tube extends to the end of the microfluidic chip and is connected to a negative pressure hole. The output tube is connected to several second reversing valves. The culture layer includes several culture chambers. The second reversing valves are connected to the culture chambers respectively. The culture chambers are all provided with nutrient holes and output holes. The culture chambers are all equipped with 3D collagen scaffolds and microelectrode arrays.

[0024] The beneficial effects of the basic solution are: the dynamic detection layer, through the design of the detection chamber and impedance electrodes, uses fluorescence detection technology and impedance flow detection technology to analyze the exhaustion status and metabolic activity of TILs in real time, accurately distinguish between dead cells and live cells, and provide accurate data for subsequent cell culture. The culture chamber of the culture layer is equipped with a 3D collagen scaffold and a microelectrode array to simulate the in vivo environment for cell expansion, monitor cytotoxicity in real time, and screen for highly active subpopulations. Overall, the combination of the dynamic detection layer and the culture layer realizes the integration of sorting, detection, and culture, reduces the number of operating steps and contamination risks, improves the efficiency and quality of cell sorting and culture, and provides strong support for adoptive cell immunotherapy.

[0025] Furthermore, the CD8 antibody is fixedly connected to the capture strip through protein A / G directional coupling technology, and the CD39 antibody is reversibly fixedly connected to the capture strip through photocrosslinking technology.

[0026] The beneficial effects of the basic scheme are as follows: CD8 antibodies are fixed through protein A / G directional coupling technology, which can specifically bind to the Fc segment of the antibody, so that the CD8 antibody is stably and directionally fixed on the capture strip, thereby efficiently capturing TILs expressing the CD8 antigen and improving the specificity and purity of the sorting. The CD39 antibody is reversibly fixed to the capture strip through photocrosslinking technology, which enables the CD39 antibody to be controllably released from the capture strip under specific light irradiation, facilitating subsequent cell elution and collection, reducing cell loss, and improving cell recovery rate. At the same time, the reversible fixation technology avoids damage to cell surface antigens and maintains the integrity and activity of the cells. Overall, this combination of antibody fixation methods not only improves the capture efficiency of TILs, but also enhances the flexibility of sorting and the recoverability of cells, further optimizing the sorting process.

[0027] Furthermore, the inner walls of the microfluidic chip that were not connected to the antibodies were coated with a PEG / BSA anti-adsorption coating to reduce nonspecific binding.

[0028] The basic solution offers the following benefits: the inner surface of the microfluidic chip, not linked to antibodies, is coated with a PEG / BSA anti-adsorption coating, effectively reducing nonspecific binding of cells and proteins. This not only reduces background signal and improves the accuracy of subsequent fluorescence and impedance detection, but also prevents random cell adhesion to the chip's inner surface, ensuring smooth cell flow and precise sorting. Furthermore, it reduces cell loss due to nonspecific binding, improves target cell recovery and sorting efficiency, and is crucial for ensuring the performance of the entire sorting system.

[0029] Furthermore, it also includes a sorting buffer, an enzymatic buffer and an acidic buffer;

[0030] The sorting buffer was PBS buffer containing 0.5% BSA, 2 mM EDTA, and 1% dextran, pH 7.4;

[0031] The enzymatic digestion buffer includes mild proteinase K, which is used to selectively cleave the antibody Fc fragment, leaving the cell surface antigen intact;

[0032] The acidic buffer was glycine-HCl buffer, pH 2.5.

[0033] The basic protocol offers the following benefits: 1. The sorting buffer contains 0.5% BSA, 2mM EDTA, and 1% dextran at a pH of 7.4, a comprehensive and optimally proportioned composition. This not only maintains cell viability and stability, preventing cell aggregation, but also provides a stable osmotic pressure environment, safeguarding cell integrity and activity, and minimizing cell damage during the sorting process caused by inappropriate buffer conditions.

[0034] 2. The enzymatic buffer contains mild proteinase K, which can specifically cleave the Fc segment of the antibody, achieving efficient separation of the antibody from the cells and retaining the integrity of the cell surface antigens. This facilitates subsequent cell detection and functional verification, and is conducive to further analysis of the biological characteristics of the cells.

[0035] 3. The acidic buffer is a glycine-HCl buffer at pH 2.5, which can be used to adjust the pH value of the local environment of the system to meet the acidic conditions required by the specific CD3 antibody elution step, helping to achieve precise manipulation and processing of cells and improving the flexibility and functionality of the entire sorting system.

[0036] Furthermore, it also includes a temperature control component, which includes a refrigerator and several heat sinks. The heat sinks are tightly attached to the top wall and bottom wall of the microfluidic chip respectively. The heat sinks are provided with observation windows corresponding to the internal pipes of the microfluidic chip. The heat sinks are provided with temperature sensors. The refrigerator and the temperature sensor are both connected to the controller signal.

[0037] The basic solution offers the following benefits: The temperature control component, through the tight fit of the cooler and heat sink, effectively regulates the temperature of the microfluidic chip, providing a suitable environment for cell sorting and culture, maintaining cell activity and stability. An observation window on the heat sink allows for real-time observation of the chip's internal piping. Simultaneously, a temperature sensor on the heat sink monitors the temperature in real time and provides feedback to the control system, enabling precise temperature control. This design ensures optimal temperature conditions for cell sorting and culture, improving both success rates and efficiency.

[0038] Furthermore, it also includes a pump assembly, which includes several negative pressure pumps and several air pumps. The negative pressure pump is connected to the waste liquid hole, negative pressure hole and output hole respectively, and the air pump is connected to the first reversing valve and the second reversing valve respectively. Both the negative pressure pump and the air pump are connected to the controller signal.

[0039] The basic solution has the following beneficial effects: through the coordinated operation of the negative pressure pump and air pump of the pump assembly, the liquid flow and cell movement trajectory within the microfluidic chip can be precisely controlled. The connection between the negative pressure pump and the waste liquid hole, negative pressure hole, and output hole ensures the timely discharge of waste liquid and the orderly delivery of cells, avoids the accumulation of liquid and pressure fluctuations within the chip, and maintains a stable sorting environment. The connection between the air pump and the reversing valve enables rapid switching and precise control of the liquid flow path, improving the flexibility and efficiency of sorting. This design not only enhances the stability and reliability of the system, but also reduces the need for manual operation, improves the degree of automation, and provides a strong guarantee for the efficient operation of the entire sorting system.

[0040] Furthermore, it also includes a pressure sensor, a fluorescence detection component, an impedance flow meter, an electrochemical workstation and a controller, and the pressure sensor is fixedly connected to the injection hole;

[0041] The fluorescence detection component includes a fluorescence spectrometer and FITC-labeled PD-1 antibodies. The PD-1 antibodies are added to the sorting buffer to mark the exhaustion status of TILs;

[0042] The impedance flow cytometer is electrically connected to the impedance electrode and is used to analyze cell size and metabolic activity and distinguish between dead and live cells;

[0043] The electrochemical workstation is electrically connected to the microelectrode array to monitor IFN-γ secretion and evaluate the anti-tumor activity of TILs;

[0044] The pressure sensor, fluorescence spectrometer, impedance flow meter and electrochemical workstation are all connected to the controller signal.

[0045] The beneficial effects of the basic program are: BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 1 is an axonometric view of the microfluidic chip in an embodiment of the present invention.

[0047] Figure 2 It is a top cross-sectional view of the pre-sorting chamber in an embodiment of the present invention.

[0048] Figure 3 2 is a top cross-sectional view of the detection chamber in an embodiment of the present invention.

[0049] Figure 4 2 is a side cross-sectional view of a culture chamber in an embodiment of the present invention.

[0050] Figure 5 for Figure 2 A magnified view of center.

[0051] Figure 6 Schematic diagram of the function of the controller in an embodiment of the present invention.

[0052] The figure marks in the drawings of the specification include: 1. microfluidic chip; 2. first reversing valve; 3. nutrient hole; 4. negative pressure hole; 5. output hole; 6. second reversing valve; 7. injection hole; 8. liquid injection hole; 9. liquid injection chamber; 10. "Y"-shaped tube; 11. delay channel; 12. pre-sorting chamber; 13. capture tube; 14. culture chamber; 15. microelectrode array; 16. microcolumn; 17. injection chamber; 18. connecting tube; 19. detection chamber; 20. output tube; 21. impedance electrode; 22. waste liquid hole; 23. 3D collagen scaffold; 24. capture strip. DETAILED DESCRIPTION

[0053] The following is further described in detail through specific implementation methods:

[0054] Example 1

[0055] Basically as attached Figure 1 、 Figure 2 、 Figure 3 、 Figure 4 and Figure 5 As shown: A tumor-infiltrating lymphocyte sorting system based on negative pressure microfluidics, including a microfluidic chip 1 and a controller, wherein the microfluidic chip 1 is provided with a sample introduction layer, a pre-sorting layer, a capture layer, a dynamic detection layer, and a culture layer which are connected in sequence;

[0056] The sampling layer is used to access the tumor cell suspension and the sorting buffer and mix the two. The sampling layer includes an injection hole 7 opened on the microfluidic chip 1. The injection hole 7 is connected to the injection chamber 17. The injection chamber 17 is connected to the "Y"-shaped tube 10. Another branch of the "Y"-shaped tube 10 is connected to the injection chamber 9. The injection chamber 9 is connected to the injection hole 8. The trunk of the "Y"-shaped tube 10 is connected to the tortuous delay channel 11.

[0057] The pre-sorting layer is used to pre-sort TILs using CD3 antibodies. The pre-sorting layer includes a pre-sorting chamber 12, which is connected to the delay channel 11. Several microcolumns 16 are bonded to the inner wall of the pre-sorting chamber 12, and the microcolumns 16 are coated with a polydopamine coating. CD3 antibodies are covalently fixed to the polydopamine coating. The other side of the pre-sorting chamber 12 is connected to a pre-sorting tube, and the middle of the pre-sorting tube is connected to a first reversing valve 2, and the first reversing valve 2 is connected to a waste liquid hole 22.

[0058] The capture layer is used to use CD8 antibodies and CD39 antibodies to combine and sort pre-sorted tumor-infiltrating lymphocytes. The capture layer includes several capture tubes 13 that are spirally wound with each other. The capture tubes 13 are all connected to the pre-sorting tube. The capture tubes 13 are interconnected by several inclined connecting tubes 18, and the inclined directions of the connecting tubes 18 are all toward the dynamic detection layer. The inner walls of the capture tubes 13 are spirally bonded with capture strips 24 made of nanofibers. Different capture antibodies are fixed on the capture strips 24, including CD8 antibodies and CD39 antibodies. The CD8 antibody is fixedly connected to the capture strip 24 through protein A / G directional coupling technology, and the CD39 antibody is reversibly fixedly connected to the capture strip 24 through photocrosslinking technology.

[0059] The dynamic detection layer is used to detect the TIL depletion status and TIL metabolic activity using fluorescence detection technology and impedance flow detection technology. The dynamic detection layer includes a detection chamber 19. One side of the detection chamber 19 is connected to the capture tube 13, and the other side of the detection chamber 19 is connected to the output tube 20. The impedance electrode 21 is symmetrically fixedly connected to the connection between the detection chamber 19 and the output tube 20.

[0060] The culture layer is used to directly access the pre-sorted TIL for direct culture. The other end of the output tube 20 extends to the end of the microfluidic chip 1 and is connected to the negative pressure hole 4. The output tube 20 is connected to several second reversing valves 6. The culture layer includes several culture chambers 14. The second reversing valves 6 are respectively connected to the culture chambers 14. The culture chambers 14 are all provided with nutrient holes 3 and output holes 5. A 3D collagen scaffold 23 and a microelectrode array 15 are installed in the culture chambers 14.

[0061] The specific implementation process is as follows: In order to solve the problem that the existing sorting technology is difficult to balance cell activity and sorting purity, and the degree of automation and reproducibility are poor, this system designed a microfluidic chip 1, which has a multi-layer structure inside, including a sampling layer, a pre-sorting layer, a capture layer, a dynamic detection layer and a culture layer, and each layer is connected in sequence to form an efficient cell sorting and culture system.

[0062] The injection chamber 17 and the injection chamber 9 achieve gentle mixing of the tumor cell suspension and the sorting buffer through the "Y"-shaped tube 10 and the tortuous delay channel 11. This structural design effectively reduces the mechanical damage that the cells may suffer during the injection process, ensures the activity and integrity of the cells entering the subsequent sorting process, and lays a good foundation for the entire sorting process. Figure 2 shown.

[0063] Pre-sorting chamber 12 utilizes a polydopamine coating on the surface of micropillars 16 to covalently immobilize CD3 antibodies. As the cell suspension flows through pre-sorting chamber 12, tumor-infiltrating lymphocytes (TILs) expressing the CD3 antigen are specifically captured, achieving a preliminary enrichment of the target cells. This process not only effectively removes most non-TIL cells, reducing the burden of subsequent sorting steps, but also significantly improves the efficiency of the entire sorting process and the purity of the target cells.

[0064] like Figure 2 As shown, the spirally wound capture tube 13 and the inclined connecting tube 18 not only extend the residence time of cells in the capture zone, but also increase the contact opportunities between cells and capture antibodies. The CD8 antibody on the capture strip 24 is fixed by protein A / G directional coupling technology, which can accurately identify and bind to TIL subpopulations expressing CD8 antigens, while the CD39 antibody is reversibly fixed by photocrosslinking technology, which makes it possible to accurately release and collect CD39-positive cells under specific conditions. This clever use of different antibody fixation methods not only ensures the specificity of capture, but also facilitates subsequent cell elution and collection, achieving efficient capture and flexible processing of target TIL subpopulations, such as Figure 5 shown.

[0065] The detection chamber 19 integrates fluorescence detection technology and impedance flow detection technology. The fluorescence spectrometer can intuitively detect the exhaustion state of TILs, providing key information for subsequent cell function evaluation. At the same time, the impedance electrode 21 monitors the size and metabolic activity of cells in real time, accurately distinguishing between dead cells and living cells, further ensuring the quality of sorted cells. The detection results of this layer provide important data support for the entire sorting process, helping to adjust the sorting parameters in real time and optimize the sorting effect, such as Figure 3 shown.

[0066] The culture chamber 14 provides a culture space for the sorted TILs that simulates the in vivo environment. The 3D collagen scaffold 23 and the microelectrode array 15 are not only conducive to cell adhesion and growth, but also can monitor cell activity and function in real time, especially the monitoring of IFN-γ secretion, which can directly reflect the anti-tumor activity of TILs. This layer design enables the sorted TILs to rapidly proliferate in an environment close to physiological conditions while maintaining their functional activity, preparing for subsequent immunotherapy applications, such as Figure 4 shown.

[0067] Overall, this system, through the careful design and coordinated operation of its components, achieves efficient sorting, detection, and culture of tumor-infiltrating lymphocytes. It not only reduces cell damage and improves sorting purity and recovery, but also provides high-quality TIL cell populations for adoptive cellular immunotherapy, demonstrating its significant clinical application value and broad prospects.

[0068] Example 2

[0069] The difference from the above embodiment is that, Figure 6 As shown: the inner wall of the microfluidic chip 1 not connected to the antibody is coated with a PEG / BSA anti-adsorption coating to reduce nonspecific binding. The tumor-infiltrating lymphocyte sorting system based on negative pressure microfluidics also includes a sorting buffer, an enzymatic hydrolysis buffer and an acidic buffer;

[0070] The sorting buffer was PBS (pH 7.4) containing 0.5% BSA, 2 mM EDTA, and 1% dextran. The enzymatic digestion buffer included mild proteinase K, which selectively cleaves the Fc region of the antibody, leaving the cell surface antigen intact. The acidic buffer was glycine-HCl buffer (pH 2.5).

[0071] It also includes a temperature control component, which includes a refrigerator and several heat sinks. The heat sinks are tightly attached to the top and bottom walls of the microfluidic chip 1 respectively. The heat sinks are provided with observation windows corresponding to the internal pipes of the microfluidic chip 1. The heat sinks are provided with temperature sensors. Both the refrigerator and the temperature sensor are connected to the controller signal.

[0072] It also includes a pump assembly, which includes several negative pressure pumps and several air pumps. The negative pressure pump is connected to the waste liquid hole 22, the negative pressure hole 4 and the output hole 5 respectively, and the air pump is connected to the first reversing valve 2 and the second reversing valve 6 respectively. The negative pressure pump and the air pump are both connected to the controller signal.

[0073] It also includes a pressure sensor, a fluorescence detection component, an impedance flow meter, an electrochemical workstation and a controller. The pressure sensor is fixedly connected to the injection hole 7; the fluorescence detection component includes a fluorescence spectrometer and FITC-labeled PD-1 antibody, and the PD-1 antibody is added to the sorting buffer to mark the exhaustion state of TIL; the impedance flow meter is electrically connected to the impedance electrode 21 for analyzing cell size and metabolic activity and distinguishing dead cells from living cells; the electrochemical workstation is electrically connected to the microelectrode array 15 for monitoring IFN-γ secretion and evaluating the anti-tumor activity of TIL; the pressure sensor, fluorescence spectrometer, impedance flow meter and electrochemical workstation are all connected to the controller signal.

[0074] The specific experimental process is as follows: 1. Experimental materials and equipment

[0075] (1) Sample: Single-cell suspension of digested solid tumor tissue (such as melanoma or ovarian cancer).

[0076] (2) Reagents: Sorting buffer: PBS (pH 7.4) containing 0.5% BSA, 2 mM EDTA, and 1% dextran.

[0077] Enzyme digestion buffer: 2 U / mL mild proteinase K, 50 mM Tris-HCl, 150 mM NaCl (pH 8.0).

[0078] Acidic buffer: glycine-HCl (pH 2.5).

[0079] FITC-labeled PD-1 antibody and anti-CD3 / CD8 / CD39 antibodies.

[0080] (3) Equipment:

[0081] Negative pressure microfluidics-based sorting system (including microfluidic chip 1, temperature control component, pump component, fluorescence detector, impedance flow instrument, electrochemical workstation); flow cytometer, ELISA kit, trypan blue staining reagent.

[0082] 2. Experimental steps

[0083] (1) Sample preparation

[0084] Tumor tissue digestion: solid tumor tissue was minced and digested with collagenase IV (1 mg / mL) and DNase I (50 U / mL) at 37°C for 1 hour, and single-cell suspension was obtained after filtration.

[0085] Cell counting: Use a hemocytometer to adjust the cell density to 1 × 10 6 cells / mL.

[0086] (2) Microfluidic chip 1 sorting

[0087] Injection and mixing: The cell suspension and sorting buffer were mixed at a volume ratio of 1:1 through a Y-shaped tube 10. The mixture passed through a tortuous delay channel 11 (flow rate 1 μL / min) to reduce shear damage.

[0088] Pre-sorting (CD3 enrichment): The mixed solution enters the pre-sorting chamber 12, where CD3 antibodies on the surface of the microcolumns 16 capture T cells. Unbound cells are discharged into the waste port 22 through the first reversing valve 2.

[0089] Capture layer sorting (CD8 / CD39 subsets): The pre-sorted cells are eluted with glycine-HCl buffer to neutralize the acidity and then enter the spiral capture tube 13, where CD8 antibodies and CD39 antibodies capture the target subsets respectively.

[0090] Dynamic detection and sorting: Fluorescence detection: FITC-PD-1 labeled exhausted T cells (excitation wavelength 488nm, emission wavelength 530nm); impedance flow cytometry: screening for viable cells with a diameter greater than 10μm and an impedance change rate greater than 15%. Negative pressure is used to control the flow rate (1-5μL / min), prioritizing cells with moderate CD39 expression.

[0091] Dissociation and collection: Target TILs are eluted by light control or proteinase K, neutralized and collected.

[0092] Culture and expansion: The sorted TILs were introduced into the 3D collagen scaffold 23 culture chamber 14 (RPMI-1640 culture medium containing 10% FBS). The microelectrode array 15 monitored the IFN-γ secretion in real time (recorded every 24 hours).

[0093] (3) Functional Verification

[0094] Flow cytometry was used to detect the retention rate of CD3 / CD8 / CD39 surface antigens, trypan blue staining was used to evaluate cell viability, and ELISA was used to quantify the levels of IFN-γ and granzyme B in the culture supernatant. The cells were co-cultured with tumor cells (ratio 1:5), and the LDH release rate was detected after 48 hours.

[0095] 3. Experimental Results

[0096] The results are shown in Table 1 below:

[0097] Table 1. Sorting efficiency and cell viability

[0098]

[0099] This system shortens sorting time from 70 days to 3 days, significantly improving TIL recovery and subset purity. Cell viability exceeds 90%, and IFN-γ secretion increases by more than 2-fold, indicating improved functional integrity. The integrated sorting and culture system reduces contamination risk, and the fully closed-loop operation minimizes errors caused by human intervention.

[0100] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus.

[0101] The above is only an embodiment of the present invention. Common knowledge such as the known specific structures and characteristics in the scheme is not described in detail here. Ordinary technicians in the field are aware of all common technical knowledge in the technical field of the invention before the application date or priority date, can obtain all existing technologies in the field, and have the ability to apply conventional experimental means before that date. Ordinary technicians in the field can improve and implement this scheme in combination with their own abilities under the inspiration given by this application. Some typical known structures or known methods should not become obstacles for ordinary technicians in the field to implement this application. It should be pointed out that for those skilled in the art, without departing from the structure of the present invention, several variations and improvements can be made, which should also be regarded as the scope of protection of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A tumor-infiltrating lymphocyte sorting system based on negative pressure microfluidics, comprising a microfluidic chip (1), characterized in that: The microfluidic chip (1) further comprises a controller, and a sample introduction layer, a pre-sorting layer, a capture layer, a dynamic detection layer and a culture layer are sequentially connected. The injection layer is used to access the tumor cell suspension and sorting buffer and mix the two; The pre-sorting layer is used to pre-sort TILs using CD3 antibodies; The capture layer is used to sort pre-sorted tumor-infiltrating lymphocytes using a combination of CD8 and CD39 antibodies; The dynamic detection layer is used to detect TIL exhaustion status and TIL metabolic activity using fluorescence detection technology and impedance flow detection technology; The culture layer is used to directly access pre-sorted TILs for direct culture.

2. The tumor-infiltrating lymphocyte sorting system based on negative pressure microfluidics according to claim 1, characterized in that: The sample injection layer includes an injection hole (7) opened on the microfluidic chip (1), the injection hole (7) is connected to an injection chamber (17), the injection chamber (17) is connected to a "Y"-shaped tube (10), another branch of the "Y"-shaped tube (10) is connected to an injection chamber (9), the injection chamber (9) is connected to the injection hole (8), and the main trunk of the "Y"-shaped tube (10) is connected to a tortuous delay channel (11); The pre-sorting layer includes a pre-sorting chamber (12), the pre-sorting chamber (12) is connected to the delay channel (11), a plurality of microcolumns (16) are fixedly connected to the inner wall of the pre-sorting chamber (12), the microcolumns (16) are coated with a polydopamine coating, and CD3 antibodies are covalently fixedly connected to the polydopamine coating. The other side of the pre-sorting chamber (12) is connected to a pre-sorting tube, the middle of the pre-sorting tube is connected to a first reversing valve (2), and the first reversing valve (2) is connected to a waste liquid hole (22).

3. The tumor-infiltrating lymphocyte sorting system based on negative pressure microfluidics according to claim 1, characterized in that: The capture layer includes a plurality of capture tubes (13) spirally wound around each other, the capture tubes (13) are all connected to the pre-sorting tube, the capture tubes (13) are mutually connected by a plurality of inclined connecting tubes (18), the inclined directions of the connecting tubes (18) are all toward the dynamic detection layer, and the inner walls of the capture tubes (13) are spirally fixed with capture strips (24) made of nanofibers, and different capture antibodies are respectively fixed on the capture strips (24), and the capture antibodies include CD8 antibodies and CD39 antibodies.

4. The tumor-infiltrating lymphocyte sorting system based on negative pressure microfluidics according to claim 1, characterized in that: The dynamic detection layer includes a detection chamber (19), one side of the detection chamber (19) is connected to the capture tube (13), the other side of the detection chamber (19) is connected to the output tube (20), and the impedance electrode (21) is symmetrically fixedly connected to the connection point between the detection chamber (19) and the output tube (20); The other end of the output tube (20) extends to the end of the microfluidic chip (1) and is connected to a negative pressure hole (4). The output tube (20) is connected to a plurality of second reversing valves (6). The culture layer includes a plurality of culture chambers (14). The second reversing valves (6) are respectively connected to the culture chambers (14). The culture chambers (14) are each provided with a nutrient hole (3) and an output hole (5). A 3D collagen scaffold (23) and a microelectrode array (15) are each provided in the culture chambers (14).

5. The tumor-infiltrating lymphocyte sorting system based on negative pressure microfluidics according to claim 4, characterized in that: The CD8 antibody is fixedly connected to the capture strip (24) through protein A / G directional coupling technology, and the CD39 antibody is reversibly fixedly connected to the capture strip (24) through photocrosslinking technology.

6. The tumor-infiltrating lymphocyte sorting system based on negative pressure microfluidics according to claim 1, characterized in that: The inner walls of the microfluidic chip (1) not connected to the antibody are coated with a PEG / BSA anti-adsorption coating to reduce nonspecific binding.

7. The tumor-infiltrating lymphocyte sorting system based on negative pressure microfluidics according to claim 1, characterized in that: It also includes sorting buffer, enzymatic buffer and acid buffer; The sorting buffer was PBS buffer containing 0.5% BSA, 2 mM EDTA, and 1% dextran, pH 7.4; The enzymatic digestion buffer includes mild proteinase K, which is used to selectively cleave the antibody Fc fragment, leaving the cell surface antigen intact; The acidic buffer was glycine-HCl buffer, pH 2.

5.

8. The tumor-infiltrating lymphocyte sorting system based on negative pressure microfluidics according to claim 1, characterized in that: The device also includes a temperature control component, which includes a refrigerator and a plurality of heat sinks. The heat sinks are respectively in close contact with the top wall and the bottom wall of the microfluidic chip (1). The heat sinks are provided with observation windows corresponding to the internal pipes of the microfluidic chip (1). The heat sinks are provided with temperature sensors. The refrigerator and the temperature sensors are both connected to the controller signal.

9. The tumor-infiltrating lymphocyte sorting system based on negative pressure microfluidics according to claim 1, characterized in that: The invention also includes a pump assembly, which includes a plurality of negative pressure pumps and a plurality of air pumps. The negative pressure pumps are respectively connected to the waste liquid hole (22), the negative pressure hole (4) and the output hole (5). The air pumps are respectively connected to the first reversing valve (2) and the second reversing valve (6). Both the negative pressure pumps and the air pumps are connected to the controller signal.

10. The tumor-infiltrating lymphocyte sorting system based on negative pressure microfluidics according to claim 1, characterized in that: It also includes a pressure sensor, a fluorescence detection component, an impedance flow meter, an electrochemical workstation and a controller, and the pressure sensor is fixedly connected to the injection hole (7); The fluorescence detection component includes a fluorescence spectrometer and FITC-labeled PD-1 antibodies. The PD-1 antibodies are added to the sorting buffer to mark the exhaustion status of TILs; The impedance flow meter is electrically connected to the impedance electrode (21) and is used to analyze cell size and metabolic activity and distinguish between dead cells and living cells; The electrochemical workstation was electrically connected to the microelectrode array (15) to monitor the secretion of IFN-γ and evaluate the anti-tumor activity of TIL; The pressure sensor, fluorescence spectrometer, impedance flow meter and electrochemical workstation are all connected to the controller signal.

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