Method for full-automatic lossless separation and enrichment of rare cells

Abstract

The invention relates to a method for full-automatic lossless separation and enrichment of rare cells, which utilizes a micro-flow device, a pressure sensor and a full-automatic control system from bottom to top, and the device can realize efficient separation and enrichment of rare cells without pre-treating thick cell liquid. By accurately controlling fluid dynamic conditions and optimizing the cell flow velocity and pressure, the method can maintain the cell activity and integrity and improve the capture rate and purity of rare cells at the same time. The design of the device allows separated and enriched rare cells to be directly collected into a collection bottle containing a cell culture medium. By means of the design, it is ensured that cells are in a proper growth environment, follow-up cell culture, downstream analysis and treatment are facilitated, and therefore the experiment efficiency and the result reliability are improved. According to the method, the traditional process is simplified, the sample treatment time is shortened, and an efficient and reliable solution is provided for cytological research and clinical diagnosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical devices and relates to a method for fully automatic and non-destructive separation and enrichment of rare cells. Background Art

[0002] Cell separation technology is a key step in biomedical research and clinical diagnosis, especially in the detection and analysis of rare cells such as circulating tumor cells (CTCs). Existing cell separation methods are mainly divided into two categories: separation methods based on cell surface-specific antigen characteristics and separation methods based on cell physical properties. Separation methods based on cell surface-specific antigen characteristics: This type of method usually relies on antigen-antibody binding and achieves cell separation through technologies such as immunomagnetic beads and flow cytometry. Although these methods have good stability, they require specific antibodies for cell surface antigens, are complex to operate, and are expensive. Separation methods based on cell physical properties: This type of method includes separation technologies based on physical properties such as cell size, mechanical properties, and electrical properties, such as filtration, centrifugation, and dielectrophoresis. These methods are relatively simple to operate and have high throughput, but improving sensitivity is a key challenge because the physical properties of cells vary very little.

[0003] In the microporous membrane filtration method based on cell size differences, filtering through a microporous membrane of a certain size combined with cell size differences is a commonly used cell sorting technology. For example, the diameter of circulating tumor cells (CTCs) is approximately 14-26 μm, while the diameters of red blood cells and white blood cells are smaller and can be filtered through a microporous membrane with a diameter of 8 μm, thereby achieving CTC enrichment. However, this method fails to fully consider the pressure difference caused by cell clogging during the sorting process, which can easily cause cell loss and damage to cell structure, affecting cell activity, resulting in poor efficiency and sensitivity of cell sorting.

[0004] In addition, existing cell filtration membrane technologies based on physical methods usually adopt a top-down flow direction. This design can easily lead to cell membrane clogging, affecting separation efficiency and cell activity. Summary of the Invention

[0005] The present invention provides a fully automatic non-destructive method for separating and enriching cells. Compared with the existing technology, this method adopts a microfluidic flow direction from bottom to top, utilizes the combination of a microfluidic chip and a cell filter membrane, and optimizes the cell flow rate and pressure by precisely controlling the fluid dynamics conditions, thereby achieving efficient separation and enrichment of rare cells. In addition, this method integrates a pressure sensor at the rear end of the microfluidic chip and the cell filter membrane for real-time monitoring of the blockage of the cell membrane. Once blockage is detected, the system immediately performs a backflush operation to prevent the liquid from being blocked, thereby improving the efficiency and accuracy of separation and enrichment. This method not only avoids the problem of cell membrane blockage, but also can directly enrich cell culture fluid, facilitates cell culture and downstream analysis, simplifies traditional processes, reduces sample processing time, and provides an efficient and reliable solution for cytological research and clinical diagnosis.

[0006] The technical solution adopted in the present invention is:

[0007] A fully automatic non-destructive separation and enrichment device for rare cells comprises a sample bottle, a sample switching valve, a collection switching valve, a collection bottle, a microfluidic chip, a pressure sensor, a three-way pipeline switching valve, a syringe, a sheath fluid bottle, a waste liquid bottle and an intelligent control system.

[0008] Furthermore, the microfluidic chip includes an upper module for outputting liquid and a lower module for inputting liquid. A chamber is formed in the middle of the upper module and the lower module, a single-cell filtration membrane is arranged in the chamber, an output flow channel connected to the chamber is arranged in the upper module, and an input flow channel connected to the chamber is arranged in the lower module. The output flow channel, the chamber and the input flow channel form a unidirectional flow path for the liquid from bottom to top.

[0009] Furthermore, the upper module includes an output layer and an upper interlayer connected to the bottom of the output layer, and the lower module includes an input layer and a lower interlayer connected to the top of the input layer. The upper interlayer and the lower interlayer are tightly combined to fix the single cell filtration membrane.

[0010] Furthermore, the single cell filtration membrane includes an inner ring and an outer ring arranged outside the inner ring, the outer ring is clamped by the upper interlayer and the lower interlayer, and the diameter of the inner ring is equal to the diameter of the chamber.

[0011] Furthermore, the thickness of the single cell filtration membrane is 5-25 μm, the diameter is 10-20 mm, and the filtration pores in the inner circle have a diameter of 5-10 μm, a pore gap of 20-30 μm, and a pore number of 150,000-300,000.

[0012] Furthermore, the output channel is arranged at the bottom of the output layer, the top of the output layer is provided with an output port connected to the output channel, the input channel is arranged at the top of the input layer, the bottom of the input layer is provided with an input port connected to the input channel.

[0013] Furthermore, the chamber is formed from bottom to top by a first cavity arranged at the top of the input layer, a second cavity arranged at the lower interlayer, a third cavity arranged at the upper interlayer, and a fourth cavity arranged at the bottom of the output layer. The first cavity, the second cavity, the third cavity and the fourth cavity are coaxially arranged and have equal diameters.

[0014] Furthermore, the output layer and the input layer are made of hard transparent polymer or thermosetting transparent polymer, and the upper interlayer and the lower interlayer are made of flexible polymer.

[0015] Furthermore, the sample bottle is connected to the A end of the sample switching valve through a transparent water pipe, the sheath liquid bottle is respectively connected to the B end of the sample switching valve and the B end of the three-way pipeline switching valve through a transparent water pipe, the C end of the sample switching valve is connected to the B end of the collection switching valve through a transparent water pipe, the collection bottle is connected to the A end of the sample collection switching valve through a transparent water pipe, the C end of the collection switching valve is connected to the input port of the microfluidic chip through a transparent water pipe, the output port of the microfluidic chip is connected to the A end of the three-way pipeline switching valve through a transparent water pipe, the C end of the three-way pipeline switching valve is connected to the syringe, and the D end of the three-way pipeline switching valve is connected to the waste liquid bottle.

[0016] Furthermore, the transparent water tube in the device has an outer diameter of 3±1 mm and an inner diameter of 2±1 mm, and the capacity of the syringe is 15-25 mL.

[0017] Furthermore, a pressure sensor is provided in the transparent water pipe between the output port of the microfluidic chip and the A end of the three-way pipeline switching valve.

[0018] Furthermore, the sample switching valve, collection switching valve, three-phase pipeline switching valve and pressure sensor are all controlled by the intelligent control system.

[0019] A method for fully automatic and non-destructive separation and enrichment of rare cells, comprising the following steps:

[0020] S1: Preparation of thick cell liquid;

[0021] S2: separation of small-sized cells;

[0022] S3: recoil;

[0023] S4: secondary cleaning and separation process;

[0024] S5: Enrichment of large cells.

[0025] Furthermore, step S1 is specifically as follows:

[0026] Prepare 5-6 mL of concentrated cell solution containing 50 10 -60 10 Small cells, 7-8 microns in size, 50 7 -60 75-6 medium-sized highly deformable cells, approximately 8-12 μm in size, and 5-6 larger rare cells, 8-20 μm in size;

[0027] Pig blood is used in the present invention, small-sized cells are red blood cells, highly deformable cells are white blood cells, and large-sized rare cells are tumor cells. In order to anticoagulate the blood during the experiment, sodium citrate anticoagulant is added to the thickened blood.

[0028] Furthermore, step S2 is specifically as follows:

[0029] Through the intelligent control system, the AC end of the sample switching valve, the BC end of the collection switching valve, and the AC end of the three-way pipeline switching valve are connected. 5-6mL of the thick cell liquid prepared by S1 is placed in a sample bottle, and a certain amount of phosphate buffer is added to the sheath fluid bottle. Through the suction syringe, the liquid cells pass through the AC end of the sample switching valve and the BC end of the collection switching valve into the input port of the microfluidic chip. At this time, the small-sized cells pass through the single-cell filtration membrane, then pass through the pressure sensor, the output port of the microfluidic chip, and the AC end of the three-way pipeline switching valve and flow into the syringe, completing the initial separation of small-sized cells.

[0030] Furthermore, step S3 is specifically as follows:

[0031] When the pressure sensor detects a change in the liquid pressure in the transparent water tube, the intelligent control system connects the BC end of the three-way pipeline switching valve, and extracts 1 mL of phosphate buffer from the sheath fluid bottle through the suction syringe. Then, the intelligent control system connects the AC end of the three-way pipeline switching valve and pushes the liquid in the syringe back into the microfluidic chip. This process can backwash the cells blocked on the single-cell filtration membrane. After completing this process, the S2 step is started again.

[0032] Furthermore, step S4 is specifically as follows:

[0033] When all the liquid in the sample bottle is extracted, the intelligent control system connects the BC end of the sample switching valve, and extracts the phosphate buffer in the sheath liquid bottle through the suction syringe. It flows into the microfluidic chip through the BC end of the sample switching valve and the BC end of the collection switching valve for secondary cell separation and cleaning. After that, the CD end of the three-way pipeline switching valve is opened through the intelligent control system to inject the small-sized cells and phosphate buffer in the syringe into the waste liquid bottle.

[0034] Furthermore, step S5 is specifically as follows:

[0035] Through intelligent control, the BC ends of the three-way pipeline switching valve are connected, and 5 mL of phosphate buffer solution is extracted from the sheath fluid bottle through the suction syringe. Then, the AC ends of the three-way pipeline switching valve and the AC ends of the collection switching valve are connected through intelligent control. The syringe is pushed to push the phosphate buffer solution in the syringe back into the microfluidic chip, and the large-sized cells deposited at the bottom of the microfluidic chip are pushed back to the AC end of the collection switching valve and into the collection bottle, completing the enrichment of large-sized cells. At this time, 5-6 mL of rare cell culture fluid can be obtained, which is beneficial for back-end cell culture or downstream analysis.

[0036] The beneficial effects of the present invention are:

[0037] 1. Efficient separation and enrichment: The combination of microfluidic chips and cell filtration membranes achieves efficient separation and enrichment of rare cells, improving separation efficiency and cell recovery rate.

[0038] 2. Non-destructive: By precisely controlling fluid dynamics conditions and adopting a bottom-up flow direction, damage to cells is reduced, and cell activity and integrity are maintained.

[0039] 3. Prevent clogging: The integrated pressure sensor and backflush operation can monitor and prevent cell membrane clogging in real time, avoiding cell loss and equipment damage caused by clogging.

[0040] 4. Simplified operation process: The fully automatic operating system simplifies the traditional cell separation process, reduces manual operations, and reduces operation complexity and labor intensity.

[0041] 5. Improve experimental efficiency: Through automated and optimized separation processes, sample processing time is significantly shortened and the overall efficiency of the experiment is improved.

[0042] 6. Wide applicability: The device is suitable for the separation of various types of cells, especially for the separation and enrichment of rare cells such as circulating tumor cells, and has broad application prospects.

[0043] 7. Convenient for subsequent analysis: The separated cells are directly collected in a collection bottle containing cell culture medium, which is convenient for subsequent cell culture, downstream analysis and processing.

[0044] 8. Intelligent control: The intelligent control system provides precise control and monitoring, ensuring the stability and repeatability of the separation process.

[0045] 9. This device directly processes concentrated cell liquids without pre-treatment. By precisely controlling fluid dynamics conditions to optimize cell flow rate and pressure, it achieves efficient separation and enrichment of rare cells while maintaining cell activity and integrity, simplifying traditional processes, reducing sample processing time, and improving experimental efficiency and the reliability of results. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a schematic diagram of the experimental device for fully automatic non-destructive separation and enrichment of rare cells;

[0047] Figure 2 Schematic diagram of the three-dimensional structure of the microfluidic chip;

[0048] Figure 3 Schematic diagram of the front cross-section structure of the microfluidic chip;

[0049] Figure 4 Schematic diagram of the cross-sectional structure of the microfluidic chip;

[0050] Figure 5 Schematic diagram of the three-dimensional structure of the single-cell filtration membrane of the microfluidic chip;

[0051] Figure 6 Schematic diagram of the front cross-section structure of the output layer of the microfluidic chip;

[0052] Figure 7 Schematic diagram of the top view of the microfluidic chip. DETAILED DESCRIPTION

[0053] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0054] Example 1

[0055] A fully automatic non-destructive separation and enrichment device for rare cells comprises a sample bottle 1, a sample switching valve 2, a collection switching valve 3, a collection bottle 4, a microfluidic chip 5, a pressure sensor 6, a three-way pipeline switching valve 7, a syringe 8, a sheath fluid bottle 9, a waste liquid bottle 10 and an intelligent control system.

[0056] Among them, the microfluidic chip includes an upper module 51 for outputting liquid and a lower module 52 for inputting liquid. A chamber 53 is formed in the middle of the upper module 51 and the lower module 52. A single-cell filtration membrane 54 is arranged in the chamber 53. An output flow channel 55 connected to the chamber 53 is arranged in the upper module 51, and an input flow channel 56 connected to the chamber 53 is arranged in the lower module 52. The output flow channel 55, the chamber 53 and the input flow channel 56 form a unidirectional flow path for the liquid from bottom to top.

[0057] Specifically, by providing an output channel 55, a chamber 53, and an input channel 56, an inverted structure is formed with a bottom-up, unidirectional flow path for liquid. After cells are input from the bottom, they are driven by pressure and sequentially pass through the chamber 53 and the single-cell filter membrane 54. Larger target cells are intercepted by the single-cell filter membrane and remain at the bottom of the chamber 53, while smaller cells and background substances are discharged through the output channel 55. This reduces the mechanical shear force on the cells during the separation process, maintains high cell activity, and improves capture efficiency by providing the single-cell filter membrane 54. The thick mixed cell liquid flows through the microfluidic chip 5, which is equipped with a single-cell filter membrane 54. By controlling the microfluidic flow from bottom to top, gravity is used to cause larger cells to naturally fall, thereby preventing clogging of the cell membrane.

[0058] The upper module 51 includes an output layer 511 and an upper interlayer 512 connected to the bottom of the output layer 511. The lower module 52 includes an input layer 521 and a lower interlayer 522 connected to the top of the input layer 521. The upper interlayer 512 and the lower interlayer 522 are tightly combined to fix the single cell filtration membrane 54.

[0059] The single cell filtration membrane 54 includes an inner ring 541 and an outer ring 542 disposed outside the inner ring 541 . The outer ring 542 is clamped by the upper interlayer 512 and the lower interlayer 522 . The diameter of the inner ring 541 is equal to the diameter of the chamber 53 .

[0060] Specifically, by limiting the thickness, pore diameter, and pore spacing of the single-cell filter membrane 54, a high-throughput design is achieved, ensuring a high capture rate. The single-cell filter membrane 4 has a thickness of 5-25 μm and a diameter of 10-20 mm. The pore diameter of the inner ring 41 is 5-10 μm, the pore spacing is 20-30 μm, and the number of pores is 150,000-300,000. The single-cell filter membrane is used to filter biological cells. Smaller cells are directly discharged through the single-cell filter membrane, while larger cells are trapped beneath the membrane.

[0061] The output channel 55 is set at the bottom of the output layer 511, and the top of the output layer 511 is provided with an output port 58 connected to the output channel 55. The input channel 56 is set at the top of the input layer 521, and the bottom of the input layer 521 is provided with an input port 57 connected to the input channel 56.

[0062] The chamber 53 is formed from bottom to top by a first cavity 531 arranged at the top of the input layer 521, a second cavity 532 arranged at the lower interlayer 522, a third cavity 533 arranged at the upper interlayer 512, and a fourth cavity 534 arranged at the bottom of the output layer 511. The first cavity 531, the second cavity 532, the third cavity 533 and the fourth cavity 534 are coaxially arranged and have equal diameters.

[0063] Specifically, cells are input from the input port 57 and flow into the first chamber 531 through the input channel 56. Under the pressure of the liquid, they flow into the second chamber 532 and the single cell filter membrane 54 from bottom to top. The captured cells will remain in the first chamber 531 and the second chamber 532. The cells that are not captured will continue to flow upward through the third chamber 533 and the fourth chamber 534, pass through the output channel 55, and be discharged from the output port 58.

[0064] The output layer 511 and the input layer 521 are made of hard transparent polymer or thermosetting transparent polymer, and the upper interlayer 512 and the lower interlayer 522 are made of flexible polymer.

[0065] Sample bottle 1 is connected to end A of sample switching valve 2 via a transparent water tube. Sheath fluid bottle 9 is connected to end B of sample switching valve 2 and end B of three-way pipe switching valve 7 via transparent water tubes. End C of sample switching valve 2 is connected to end B of collection switching valve 3 via a transparent water tube. Collection bottle 4 is connected to end A of sample and collection switching valve 3 via a transparent water tube. End C of collection switching valve 3 is connected to input port 57 of the microfluidic chip via a transparent water tube. Output port 58 of the microfluidic chip is connected to end A of three-way pipe switching valve 7 via a transparent water tube. End C of three-way pipe switching valve 7 is connected to syringe 8, and end D of three-way pipe switching valve 7 is connected to waste liquid bottle 10. After separation and enrichment, rare cells are directly collected into collection bottle 4 containing cell culture medium through collection switching valve 3, ensuring that the cells are in a suitable growth environment for subsequent cell culture, downstream analysis, and processing.

[0066] The transparent water tube in the device has an outer diameter of 3±1 mm and an inner diameter of 2±1 mm. The capacity of the syringe 8 is 15-25 mL, which can effectively reduce the resistance in the pipeline, accurately control the flow rate of the fluid and facilitate observation.

[0067] Among them, a pressure sensor 6 is installed in the transparent water pipe between the output port 58 of the microfluidic chip and the A end of the three-way pipeline switching valve 7. The pressure sensor 6 is used to monitor the blockage of the cell filter membrane in real time. Once blockage is detected due to smaller cells or larger deformed cells, the system immediately performs a backflush operation to prevent the liquid from being blocked, thereby improving the efficiency and accuracy of separation and enrichment.

[0068] The sample switching valve 2, the collection switching valve 3, the three-phase pipeline switching valve 7 and the pressure sensor 6 are all controlled by the intelligent control system to realize the functions of fully automatic detection, pre-experiment cleaning, cell separation, backflush collection and post-experiment cleaning.

[0069] Example 2

[0070] A method for fully automatic non-destructive separation and enrichment of rare cells, based on the fully automatic non-destructive separation and enrichment device for rare cells in Example 1, comprises the following steps:

[0071] S1: Preparation of thick cell liquid

[0072] Prepare 5-6 mL of concentrated cell solution containing 50 10 -60 10 Small cells, 7-8 microns in size, 50 7 -60 7 5-6 medium-sized highly deformable cells, approximately 8-12 μm in size, and 5-6 larger rare cells, 8-20 μm in size;

[0073] Pig blood is used in the present invention. Small-sized cells are red blood cells, highly deformable cells are white blood cells, and large-sized rare cells are tumor cells. In order to anticoagulate the blood during the experiment, sodium citrate anticoagulant is added to the thickened blood.

[0074] S2: Isolation of small cells

[0075] The AC end of the sample switching valve 2, the BC end of the collection switching valve 3, and the AC end of the three-way pipeline switching valve 8 are connected through the intelligent control system. 5-6 mL of the thick cell liquid prepared in S1 is placed in the sample bottle 1. A certain amount of phosphate buffer is added to the sheath fluid bottle 9. By suctioning the syringe 8, the liquid cells pass through the AC end of the sample switching valve 2 and the BC end of the collection switching valve 3 and flow into the input port 57 of the microfluidic chip 5. At this time, the small-sized cells pass through the single-cell filter membrane 54, then pass through the pressure sensor 6, the output port 58 of the microfluidic chip 5, and the AC end of the three-way pipeline switching valve 8 before flowing into the syringe 8, completing the initial separation of small-sized cells;

[0076] S3: Recoil

[0077] When the pressure sensor 7 detects a change in the liquid pressure in the transparent water tube, the intelligent control system connects the BC end of the three-way pipeline switching valve 7 and extracts 1 mL of phosphate buffer from the sheath fluid bottle 9 through the suction syringe 8. The intelligent control system then connects the AC end of the three-way pipeline switching valve 7 and pushes the liquid in the syringe 8 back into the microfluidic chip 5. This process can backwash cells blocked on the single-cell filter membrane 54. After this process is completed, step S2 is started again;

[0078] S4: Secondary cleaning and separation process:

[0079] When all the liquid in the sample bottle 1 is completely extracted, the intelligent control system connects the BC end of the sample switching valve 2, and extracts the phosphate buffer in the sheath liquid bottle 9 through the suction syringe 8. The phosphate buffer flows into the microfluidic chip 5 through the BC end of the sample switching valve 2 and the BC end of the collection switching valve 3 to perform a secondary cell separation and washing process. After that, the CD end of the three-way pipeline switching valve 8 is opened by the intelligent control system, and the small-sized cells and phosphate buffer in the syringe 8 are injected into the waste liquid bottle 11.

[0080] S5: Enrichment of large cells

[0081] Through intelligent manipulation, the BC ends of the three-way pipeline switching valve 8 are connected, and 5 mL of phosphate buffer in the sheath fluid bottle 9 is extracted through the suction syringe 8. Then, the AC ends of the three-way pipeline switching valve 8 and the AC ends of the collection switching valve 3 are connected through intelligent manipulation, and the syringe 8 is pushed to push the phosphate buffer in the syringe 8 back into the microfluidic chip 5. The large-sized cells deposited at the bottom of the microfluidic chip are pushed back to the AC end of the collection switching valve 3 and enter the collection bottle 4 to complete the enrichment of the large-sized cells. At this time, 5-6 mL of rare cell culture fluid can be obtained, which is beneficial to the back-end cell culture or downstream analysis.

[0082] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention are included in the scope of protection of the present invention.

Claims

1. A fully automatic non-destructive method for separating and enriching rare cells, characterized in that: The steps include: S1: Preparation of thick cell liquid; S2: separation of small-sized cells; S3: recoil; S4: secondary cleaning and separation process; S5: Enrichment of large cells.

Images