Centrifugal separation mechanism and tumor cell separation device

Abstract

The invention provides a centrifugal separation mechanism and a tumor cell separation device. The centrifugal separation mechanism comprises a first bottle body used for containing liquid to be separated, and the first bottle body is arranged to be in a cone shape or a circular truncated cone shape with the axis vertically arranged and is rotationally arranged around the axis; an opening of the first bottle body faces upwards and is located on the end face of the end with the smaller diameter; the side wall of the first bottle body is of a filter layer structure, and when the first bottle body is in a rotating state, part of the liquid to be separated can penetrate through the filter layer structure. By arranging the rotating conical bottle body, autoblood containing tumor cells is injected into the bottle body for centrifugal separation, pure red blood cells not containing tumor cells can be obtained, and the red blood cells can be used for intraoperative recovery type autotransfusion and are high in efficiency.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor cell separation, in particular to a centrifugal separation mechanism and a tumor cell separation device. Background Art

[0002] Malignant tumors have become an important cause of disease that seriously threatens human health. Tumor patients are in serious condition and often need to undergo surgical treatment, which will cause blood loss during surgical treatment. At present, allogeneic blood transfusion is mainly used in clinical practice. Statistics show that tumor patients account for 30% of clinical blood usage, and are veritable major blood users. Solving the blood transfusion problem for tumor patients is imperative. Tumor patients receive blood transfusions mainly to expand blood volume, increase hemoglobin, and increase oxygen content. With the increase in the number of tumor patients, the already tense blood supply situation has become more severe. At present, even with the transfusion of allogeneic blood, there is still a situation of blood supply exceeding demand, and the situation of tight clinical blood use is very serious.

[0003] In addition, although perioperative allogeneic blood transfusion in patients with malignant tumors can improve circulation and increase oxygen supply, it also brings great risks. It not only increases the rate of adverse reactions to general blood transfusions, but also increases transfusion-related immunosuppression, which increases tumor-specific complications and mortality. A large number of clinical and basic studies have shown that transfusion of allogeneic blood in patients with malignant tumors can cause suppression of the patient's immune function, leading to tumor proliferation, metastasis, and recurrence, and shortening life expectancy.

[0004] Under the conditions of blood shortage and transfusion risks, autologous blood transfusion has become an important treatment method that is indispensable for saving lives. It not only saves blood, but also effectively reduces perioperative complications of allogeneic blood transfusion. The gradual replacement of allogeneic blood transfusion by autologous blood transfusion has become an inevitable trend in the development of medicine. Autologous blood transfusion includes three types: recovery type, dilution type, and storage type. Among them, recovery autologous blood transfusion has been widely used in various surgical operations, which plays a significant role in saving blood and alleviating the increasingly tense blood use contradictions in clinical practice.

[0005] Therefore, intraoperative salvage autologous blood transfusion, as one of the important measures of "blood protection", has been widely used in orthopedic, cardiac, liver, urological, obstetric and other surgeries. However, whether it can be applied to patients undergoing malignant tumor surgery is still controversial. The focus of the debate is whether transfusing autologous blood mixed with tumor cells back to patients may lead to tumor proliferation, metastasis, and recurrence. Although a large number of clinical studies abroad have retrospectively analyzed and believed that the risk is low, there is still a lack of relevant basic research, and the mechanism is unclear. Due to concerns that salvage autologous blood transfusion in patients with malignant tumors may cause tumor cell input and lead to spread and metastasis, salvage autologous blood transfusion is listed as a contraindication in malignant tumor surgery.

[0006] Currently, there are two main research areas for separating or inactivating tumor cells from the blood: one is to collect blood from the surgical field of tumor patients through an autologous blood transfusion machine, and combine it with a leukocyte filter or ionizing radiation, with the aim of reducing or killing tumor cells and returning them to the patient to supplement red blood cells; the other is to separate circulating tumor cells in the blood through antigen-independent or antigen-dependent methods, with the main purpose of performing in vitro diagnosis of tumors by drawing a small amount of peripheral blood from the patient.

[0007] 1. Related research on intraoperative autologous blood transfusion for tumor patients

[0008] (1) Leukocyte filter

[0009] Principle: Mechanical blockage and adsorption prevent white blood cells from passing through, allowing red blood cells with strong deformability and smooth surface to pass through. In a study of liver transplantation in patients with advanced liver cancer, the results showed that after filtering every 500 ml of autologous blood with a white blood cell filter, there was no statistical difference in the recurrence rate and survival rate between patients who used autologous blood transfusion in combination with a white blood cell filter and those who did not use autologous blood transfusion. Related studies on improved white blood cell filters have shown that the improved white blood cell filter (pore size 12-17 μm) designed on the basis of conventional white blood cell filters (pore size 25-50 μm) showed cell membrane rupture and cytoplasm leakage in circulating tumor cells passing through the improved white blood cell filter. After filtering the recovered autologous blood of patients with metastatic spinal tumors with the improved white blood cell filter, the positive rate of circulating tumor cells was significantly lower than that of conventional white blood cell filters (12.5% ​​vs 60%).

[0010] (2) Ionizing radiation

[0011] Gong et al. mixed washed red blood cells from healthy volunteers with liver cancer cells HepG2, gastric cancer cells SGC7901 and colon cancer cells SW620, and then irradiated the mixed cells with 30, 50 and 100 Gy of γ-rays, respectively, and detected tumor cells and observed the effects of irradiation on tumor cells and red blood cells. The study showed that 50 Gy of γ-ray irradiation was the best irradiation intensity for liver cancer cells HepG2, which can ensure the inactivation of tumor cells without affecting the viability of red blood cells. After SW620 cells were irradiated with 50 Gy of γ-rays, cell colony formation was still observed after 14 days of culture, which may not be suitable for γ-ray irradiation inactivation. Zhang et al. conducted similar studies using 30 and 50 Gy of X-rays. The results showed that 30 Gy of X-rays was the best irradiation intensity for HepG2, Huh7 and SK-Hep1 liver cancer cells, and it did not affect the viability of red blood cells.

[0012] However, although autologous blood transfusion technology combined with leukocyte filter can reduce the number of tumor cells to a certain extent, it is still not effective when the tumor cell concentration reaches 2×10 7 / 200ml, it cannot completely filter out tumor cells, and its safety needs further verification. Ionizing radiation is not widely used in clinical practice because of its expensive equipment and operational risks.

[0013] 2. Related research on circulating tumor cell separation technology

[0014] Circulating tumor cells refer to tumor cells that fall off from the primary tumor and extravasate into the blood circulation. CTCs isolation methods are divided into antigen-dependent methods based on biological characteristics and antigen-independent methods based on physical characteristics. Among them, antigen-dependent methods are divided into epithelial circulating tumor cell markers, mesenchymal circulating tumor cell markers and cancer-specific antigens; antigen-independent methods are mainly based on the physical characteristics of tumor cells, including cell size, density, cell charge and other characteristics.

[0015] (1) Antigen-dependent method:

[0016] ① Positive separation method Most cancers are of epithelial origin, and EpCAM is a "universal" epithelial tumor marker. The Cell Search system is the only device approved by the FDA for clinical use, which uses magnetic beads coated with EpCAM antibodies to enrich circulating tumor cells.

[0017] ② Negative separation method combines anti-CD45 antibodies with magnetic beads to remove white blood cells. For example, EasySep technology uses a magnetic field to retain white blood cells, so that tumor cells that do not express CD45 are retained in the supernatant. This method does not rely on tumor-specific markers, but circulating tumor cells in the blood are a minority. During the enrichment process, circulating tumor cells are wrapped by a large number of red blood cells, which increases the difficulty of collecting circulating tumor cells.

[0018] (2) Antigen-independent method:

[0019] ① Separation of circulating tumor cells based on cell size: The size of circulating tumor cells is 14-26μm, white blood cells are 7-20μm, and red blood cells are 7-8μm. FAST developed by Clinomics allows rapid separation of circulating tumor cells with high purity from whole blood. The device consists of a track-etched polycarbonate membrane with 8μm holes, which selectively separates circulating tumor cells based on cell size. However, this method is not easy to separate larger blood cells and tumor cells, and the filtration device is prone to membrane clogging.

[0020] ② Based on cell density separation, the commonly used cell density gradient media are Ficoll and OncoQuick. The former has a narrower separation range and is mostly used to separate peripheral blood mononuclear cells; the latter has a porous sieve, which can effectively reduce leukocyte contamination. The recovery rates of Ficoll and OncoQuick are both about 70% to 90%.

[0021] ③ Fluid mechanics: This method mainly separates cells based on the different characteristics of particle movement in the fluid in the microfluidic chip. There are three main methods: deterministic lateral displacement, inertial focusing and eddy current capture technology.

[0022] ④ Based on ultrasonic enrichment technology, some researchers have used microfluidic electrophoresis chips to enrich circulating tumor cells according to the different sizes of circulating tumor cells and white blood cells. Under the action of high-frequency sound waves, circulating tumor cells move to the center of the pressure channel, while white blood cells move to the side of the channel wall. The advantages of this method are small damage to cells and strong flexibility; however, its disadvantage is that it cannot directly process whole blood and red blood cells must be removed in advance.

[0023] ⑤ Dielectrophoresis technique Electrophoresis is the process of moving charged particles through the interaction between the net charge on the charged particles and the applied electric field. Neutral particles themselves have no net charge, and dielectrophoretic force can be generated through the polarization of neutral particles in a non-uniform electric field. Therefore, circulating tumor cells can be separated and enriched by using dielectrophoretic force. Studies have shown that the dielectrophoretic field flow fractionation method can separate circulating tumor cells from clinical blood samples. This method can utilize the inherent characteristics of tumor cells, does not require labeling, and has a cell capture rate of more than 90%. However, the equipment required for dielectrophoresis technology is complex and the flux is lower than other methods, and its enrichment efficiency still needs to be further improved.

[0024] Although a lot of research has been conducted in related fields at home and abroad on the removal or inactivation of tumor cells in the blood, and the activity of tumor cells has been inhibited or their number has been reduced to a certain extent, these methods still have problems of low efficiency in terms of capture efficiency and blood flux, making them difficult to use in clinical practice. Summary of the invention

[0025] The present invention provides a centrifugal separation mechanism and a tumor cell separation device, which can solve the problem of low efficiency in the prior art in removing or inactivating tumor cells in blood.

[0026] In order to solve the above problems, the present invention provides a centrifugal separation mechanism, comprising:

[0027] A first bottle body for containing the liquid to be separated, wherein the first bottle body is configured to be in a cone or truncated cone shape with a vertical axis and is rotatably disposed about the axis; the opening of the first bottle body is configured to face upward and is located on the end surface of the end with a smaller diameter;

[0028] The side wall of the first bottle body is configured as a filter layer structure. When the first bottle body is in a rotating state, a portion of the liquid to be separated can pass through the filter layer structure.

[0029] In an optional solution of the present invention, the first bottle opening is connected to a first injection tube and a first outlet tube, the end of the first injection tube extends into the interior of the bottle, and the other part of the liquid to be separated that has not passed through the filter layer structure is discharged from the first bottle through the first outlet tube.

[0030] In an optional solution of the present invention, the filter layer structure includes two membrane layers and a filling layer located between the two membrane layers, the pore size of the membrane layer is 6-10 μm, and the filling layer includes spherical particles with an outer diameter of 10-20 μm.

[0031] In an optional solution of the present invention, the filter layer structure further comprises a mesh support structure located outside the two membrane layers, and the mesh size of the support structure is larger than the pore size of the membrane layer.

[0032] In an optional solution of the present invention, the material of the spherical particles includes polymethyl methacrylate; or / and the spherical particles are composed of particles with an outer diameter of 10 μm and 20 μm mixed in equal proportions.

[0033] In an optional solution of the present invention, the centrifugal separation mechanism also includes a collecting hood, which is covered on the side walls and bottom walls of the first bottle body and is used to collect the filtrate passing through the filter layer structure; preferably, the collecting hood is provided with a discharge port at a corresponding position of the bottom wall for discharging the filtrate.

[0034] According to another aspect of the present invention, a tumor cell separation device is provided, comprising an autologous blood flow path and the centrifugal separation mechanism as described above; the centrifugal separation mechanism is arranged in the autologous blood flow path and is used to separate tumor cells in the autologous blood.

[0035] In an optional scheme of the present invention, the tumor cell separation device also includes an inlet, a filter, a centrifuge cup and a blood storage device, and the autologous blood flow path includes the inlet, the filter, the centrifuge mechanism, the centrifuge cup and the blood storage device connected in sequence; the filtrate of the centrifuge mechanism is stored in the blood storage device after passing through the centrifuge cup.

[0036] In an optional scheme of the present invention, the centrifugal cup includes a second bottle body, which is configured to be a cone or a truncated cone with an axis vertically arranged, and is rotatably arranged about the axis; the opening of the second bottle body is arranged upward and is located on the end surface of the end with a smaller diameter; the opening of the second bottle body is connected to a second injection pipe and a second outlet pipe, the second injection pipe is connected to the interior of the second bottle body, and the filtrate is injected into the interior of the second bottle body; when the second bottle body is in a rotating state, the filtrate undergoes centrifugal stratification, and the stratified filtrate is discharged separately through the second outlet pipe.

[0037] In an optional solution of the present invention, a driving pump is provided between the filter and the first bottle body, and between the first bottle body and the second bottle body, and the driving pump includes a peristaltic pump.

[0038] The present invention has the following beneficial effects:

[0039] By setting up a rotating conical bottle, autologous blood containing tumor cells is injected into the bottle and centrifuged to obtain pure red blood cells without tumor cells. It can be used as intraoperative recovery autologous blood transfusion with high efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the specific implementation of the present invention or the technical solution in the prior art, the following briefly introduces the drawings required for use in the specific implementation or the prior art description. Obviously, the drawings described below are some implementations of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0041] Figure 1 is a schematic structural diagram of the tumor cell separation device of the present invention;

[0042] Figure 2 It is a schematic structural diagram of the first bottle body of the present invention;

[0043] Figure 3 A cross-sectional schematic diagram of a structure of the centrifugal separation mechanism of the present invention;

[0044] Figure 4 is a cross-sectional schematic diagram of another structure of the centrifugal separation mechanism of the present invention;

[0045] Figure 5 For the present invention Figure 4 Schematic diagram of local enlargement and separation principle;

[0046] Figure 6 It is a schematic cross-sectional view of the structure of the centrifugal cup of the present invention.

[0047] The reference numerals are as follows:

[0048] 1. Inlet; 2. Heparin anticoagulant; 3. Filter; 4. Negative pressure pump; 5. Physiological saline container; 6. First drive pump; 7. Centrifugal separation mechanism; 8. Tumor cell container; 9. Second drive pump; 10. Centrifugal cup; 11. Waste liquid container; 12. Blood storage device;

[0049] 71, side wall; 711, outer film layer; 712, filling layer; 713, inner film layer; 72, bottom wall; 73, first injection pipe; 74, first outlet pipe; 75, collection cover; 751, discharge port;

[0050] 101. Second bottle body; 102. Second injection pipe; 103. Second outlet pipe. DETAILED DESCRIPTION

[0051] In the description of the present invention, it is necessary to understand that if there are terms such as "center", "inside", "outside", "axial", "radial", "circumferential" and the like indicating orientation or positional relationship, unless otherwise specified, they are understood to be based on the orientation or positional relationship shown in the drawings, which is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention.

[0052] In addition, if there is a feature defined as "first" or "second", it is only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Features defined as "first" or "second" may explicitly or implicitly include at least one of the defined features. If there is a description of "plurality", the general meaning is to include at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0053] In the present invention, unless otherwise clearly specified and limited, the terms such as "installed", "connected", "connected", "fixed" and the like should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integrated connection; it can be a mechanical connection or an electrical connection, it can be a direct connection, or it can be an indirect connection through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements. For those skilled in the art, the specific meanings of the above terms in the present invention can be understood according to the specific circumstances.

[0054] In the description of this specification, if the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" appear, it means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification and the features of different embodiments or examples without contradiction.

[0055] The present invention provides a centrifugal separation mechanism, comprising:

[0056] A first bottle body for containing the liquid to be separated, wherein the first bottle body is configured to be in a cone or truncated cone shape with a vertical axis and is rotatably disposed about the axis; the opening of the first bottle body is configured to face upward and is located on the end surface of the end with a smaller diameter;

[0057] The side wall 71 of the first bottle body is configured as a filter layer structure. When the first bottle body is in a rotating state, a portion of the liquid to be separated can pass through the filter layer structure.

[0058] Figures 2 to 5 A centrifugal separation mechanism 7 according to a specific embodiment of the present invention is provided.

[0059] Figure 2 The schematic diagram of the structure of the first bottle body of the present invention is shown; the first bottle body is conical, and is surrounded by a side wall 71 and a bottom wall 72 to form a structure with an opening, and the opening is arranged at the tip of the cone and is arranged upward; wherein the side wall 71 is a filter layer structure, especially when the first bottle body is in a rotating state, so that the internal liquid to be separated is in a centrifugal motion process, which can enable the liquid to be separated in the first bottle body to partially pass through the filter layer structure to achieve centrifugal separation.

[0060] Since the side wall 71 of the first bottle body is inclined, the centrifugal speed of the first bottle body can also be changed during the rotation process, so that the force on the substance that is difficult to pass through the filter layer structure during the separation process changes, inducing it to change its position, thereby avoiding the problem of clogging of the fixed channel. In the centrifugal separation mechanism 7 of the present invention is used in the separation device for tumor cells, the substance that is difficult to pass through the filter layer structure is a tumor cell. After the tumor cells are displaced during the centrifugal separation process, the liquid in the internal channel is collected to further analyze the characteristics of the tumor cells and provide information for subsequent treatment.

[0061] The shape of the first bottle body may also be a truncated cone, with the opening being located at the center of the upper end surface.

[0062] Figure 3 A cross-sectional schematic diagram of a structure of a centrifugal separation mechanism 7 of the present invention is shown; the centrifugal separation mechanism 7 includes a first bottle body, a collecting cover 75, a first injection pipe 73 and a first outlet pipe 74, the end of the first injection pipe 73 extends into the first bottle body, and the liquid to be separated is injected into the first bottle body. As the first bottle body rotates, the liquid to be separated is centrifugally separated, and part of the filtrate that passes through the filter layer structure is collected by the outer collecting cover 75 and discharged from the centrifugal separation mechanism 7 through the outlet 751; and the liquid to be separated that does not pass through the filter layer structure moves upward as the first bottle body rotates, and is discharged from the first bottle body through the first outlet pipe 74 connected to the first bottle body.

[0063] For the first injection tube 73 and the first discharge tube 74, the two form a sleeve structure and are hermetically inserted into the opening of the first bottle body; when the first bottle body rotates, the sleeve and the inner wall at the opening are in sliding fit.

[0064] Figure 4 Fig. shows a sectional schematic view of another structure of the centrifugal separation mechanism 7 of the present invention; different from Figure 3 the centrifugal separation mechanism 7 shown, a conical or frustum-shaped structure is provided on the outer periphery of the part of the first injection tube 73 extending into the first bottle body, so that the extending part and the inner wall of the first bottle body are both arranged at intervals to form a flow channel. In this way, the liquid to be separated is in a flowing state along the flow channel in the first bottle body to achieve centrifugal separation.

[0065] Figure 5 Fig. shows an enlarged and principle schematic view of the filter layer structure of the present invention; the filter layer structure includes an outer membrane layer 711, an inner membrane layer 713, and a filling layer 712 located between the outer membrane layer 711 and the inner membrane layer 713. Both the outer membrane layer 711 and the inner membrane layer 713 are porous film structures with a pore diameter of 6 - 10 μm, and the filling layer 712 includes spherical particles with an outer diameter of 10 - 20 μm.

[0066] For the pore diameter of the above-mentioned membrane layer and the size of the spherical particles in the filling layer 712, it is to facilitate the filter layer structure to separate and process the blood containing tumor cells. Utilizing the deformation ability of red blood cells in the blood and the rigidity of nucleated tumor cells, a smaller filtration barrier is constructed by spherical particles. Under the drive of centrifugal force, pure deformable healthy red blood cells are sorted out, the tumor cells recovered from the surgical field of malignant tumor patients are removed, and the pure red blood cells are transfused back to the patients, increasing the safety and reliability of blood transfusion.

[0067] The principle of the filling layer separation is that red blood cells themselves do not have a nucleus, so they can deform and pass through the gaps between spherical particles; while nucleated tumor cells are difficult to pass through the gaps between spherical particles due to the difficulty of nuclear deformation. In this way, combined with the action of centrifugal force, red blood cells and tumor cells are separated.

[0068] For the filter layer structure, a reticular support structure can also be provided on the outer sides of the two membrane layers, and the mesh size of the support structure is larger than the pore diameter of the membrane layer.

[0069] The support structure plays a role in fixing and supporting the filter layer structure. It is equivalent to clamping the filter layer structure by the support structure, and then jointly forming the side wall 71 of the first bottle body; the mesh size of the support structure is larger, which can reduce the resistance to liquid, facilitate the liquid to contact the membrane layer and the filling layer 712 after passing through the support structure, and improve the centrifugal separation effect.

[0070] In an optional solution of the present invention, the material of the spherical particles includes polymethyl methacrylate; or / and the spherical particles are composed of particles with an outer diameter of 10 μm and 20 μm mixed in equal proportions.

[0071] For the selection of spherical particles, polymethyl methacrylate is preferably used, which can be mixed in equal proportions and then placed between two layers of membranes. A metal filter with a pore diameter of 100 μm is provided on the outside of the two filter membranes as a filter membrane support material, and a liquid inflow channel is provided between the inner metal filter and the outer wall of the first bottle body, and a liquid outflow channel is provided between the outer metal filter and the outer wall of the first bottle body. The outflow channel contains the filtrate that passes through the filter layer structure, and the filtrate for filtering tumor cells is blood that does not contain tumor cells, and the nucleated cells such as tumor cells that cannot pass through the filter layer structure are finally discharged from the first bottle body through the first outlet tube 74.

[0072] According to another aspect of the present invention, a tumor cell separation device is provided, comprising an autologous blood flow path and the centrifugal separation mechanism 7 as described above; the autologous blood contains tumor cells, and the autologous blood flow path is connected to the first bottle body through the injection tube.

[0073] Figure 1 A tumor cell separation device according to a specific embodiment of the present invention is provided.

[0074] Figure 1 The schematic diagram of the structure of the tumor cell separation device of the present invention is shown; the tumor cell separation device mainly includes an inlet 1, a heparin anticoagulant 2, a filter 3, a negative pressure pump 4, a physiological saline container 5, a first driving pump 6, a centrifugal separation mechanism 7, a tumor cell container 8, a second driving pump 9, a centrifugal cup 10, a waste liquid container 11 and a blood storage device 12; the inlet 1 is connected to the centrifugal separation mechanism 7 through the filter 3, and the filtrate after centrifugation is sent to the centrifugal cup 10 for centrifugal stratification to achieve blood concentration, water is sent to the waste liquid device, and the concentrated blood is sent to the blood storage device 12 for easy recycling.

[0075] The injection port 1 is an aspirator connector for aspirating blood flowing out during surgery; an injection port for a heparin anticoagulant 2 is also provided on the main circulation pipeline between the injection port 1 and the filter 3, and a negative pressure pump 4 is connected to the filter 3; the flow of blood is connected to a centrifugal separation mechanism 7 through a first drive pump 6, to which a physiological saline container 5 is also connected, to increase the fluidity of the blood, and the physiological saline is mixed with the blood in the main circulation pipeline and injected into the first drive pump 6 together; the first drive pump 6 injects the mixed fluid into the centrifugal separation mechanism 7, and separates it under the action of centrifugation, so that cells are forced to enter the outer pipeline from the inner pipeline of the first bottle body through the filtration barrier composed of microspheres, and cells and other substances that fail to pass through the barrier are blocked in the inner pipeline, and cells and blood that have passed through the microsphere filter plate enter the centrifugal cup 10 under the action of the second drive pump 9, and the cell components and liquid components are separated under the action of centrifugation, and the concentrated cell components are injected into the blood storage device 12, and the liquid components are injected into the waste liquid bag.

[0076] The saline solution of the heparin anticoagulant 2, more specifically the physiological saline solution of sodium heparin, is injected into the filter 3, and the pipeline therebetween is a closed pipeline without any opening of the side wall 71, and the main circulation pipeline between the injection port of the physiological saline container 5 and the first driving pump 6 is also a closed pipeline without any opening of the side wall 71; the negative pressure pump 4 is connected to the filter 3 through a pipeline, and maintains a negative pressure state in the filter 3 to ensure that the fluid inlet 1 can continuously attract fluid.

[0077] Figure 6 The schematic diagram of the structure of the centrifugal cup 10 of the present invention is shown; the centrifugal cup 10 includes a conical second bottle body 101, the second bottle body 101 is opened upward and is rotatably arranged; the second bottle body 101 opening is connected to a second injection pipe 102 and a second outlet pipe 103, the second injection pipe 102 is connected to the interior of the second bottle body 101, and filtrate is injected into the interior of the second bottle body 101; when the second bottle body 101 is in a rotating state, the filtrate undergoes centrifugal stratification, and the water precipitated in the filtrate is discharged through the second outlet pipe 103.

[0078] The tumor cell separation device of the present invention can be used to separate tumor cells from autologous blood recovered from the surgical field of a malignant tumor patient. The separation device can be manufactured and tumor cell separation can be achieved through the following process, including:

[0079] A. Prepare filter 3, open two openings, connect one opening to negative pressure pump 4 to ensure that the inside of filter 3 is in negative pressure state, and connect the other opening to sterile inlet 1 to drain blood from the surgical field. Establish a liquid pipeline at the tail end of the sterile suction device to inject anticoagulant saline to ensure the airtightness and sterility of the entire liquid circuit.

[0080] B. Both the first driving pump 6 and the second driving pump 9 are peristaltic pumps, which drive the fluid to be transported into the centrifugal cup 10 during operation, and stop driving when the liquid injected into the centrifugal cup 10 reaches the upper limit.

[0081] C. Prepare a truncated cone-shaped centrifugal separation mechanism 7. Mix polymethyl methacrylate microspheres (PMMA) with diameters of 10 μm and 20 μm in equal proportion and fill them between two layers of filter membranes with a pore size of 8 μm. Place two metal gaskets with uniform pore sizes on the outside of the filter membrane to support the filter membrane. Place the microspheres, filter membrane and metal gasket system in a truncated cone container, and make the container seamlessly connected with the metal gasket and filter membrane to ensure that the position of the microspheres is fixed and cannot be exposed. In addition, liquid can be injected between the inner metal gasket and the inner layer of the truncated cone container, and liquid can be drained between the outer metal gasket and the outer layer of the truncated cone.

[0082] D. Prepare a truncated cone-shaped centrifugal cup 10 for concentration. Drain the blood sample into the truncated cone-shaped centrifugal cup 10, start the centrifugal cup 10 to rotate, and separate the blood sample into an upper clear liquid and a lower cell sediment under the action of centrifugal force. Introduce the lower cell sediment into the blood storage 12 for recycling, and inject the upper clear liquid into the waste liquid bag.

[0083] The working process of a tumor cell separation device of the present invention is described below.

[0084] like Figure 1 The device shown is a device for separating tumor cells in blood fluid, including blood from the surgical field and anticoagulant saline, which are mixed under the action of a negative pressure suction pump and enter the filter 3 through a fluid inlet 1. The filter 3 can filter out larger gauze debris and blood clots and other substances. The filtered fluid enters the centrifugal separation mechanism 7 under the action of a first driving pump 6. Driven by centrifugal force, the filtrate flows to the outer pipeline of the centrifugal cup 10. In the process of passing through the microsphere filter plate, tumor cells and white blood cells are blocked inside the barrier. Red blood cells use their deformation ability to pass through the filter plate and enter the outer fluid channel. The cells contained in the fluid in this channel are pure red blood cells. The fluid separated by the centrifugal separation mechanism 7 enters the centrifugal cup 10 for concentration under the action of a second driving pump 9. Driven by centrifugal force, the fluid is divided into a supernatant and a pure red blood cell precipitate. The pure red blood cells are drained into the blood storage device 12, and the upper clear liquid is discharged from the waste bag.

[0085] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. Those skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A centrifugal separation mechanism, characterized in that: include: A first bottle body for containing the liquid to be separated, wherein the first bottle body is configured to be in a cone or truncated cone shape with a vertical axis and is rotatably disposed about the axis; the opening of the first bottle body is configured to face upward and is located on the end surface of the end with a smaller diameter; The side wall of the first bottle body is configured as a filter layer structure. When the first bottle body is in a rotating state, a portion of the liquid to be separated can pass through the filter layer structure.

2. The centrifugal separation mechanism according to claim 1, characterized in that: The first bottle opening is connected to a first injection tube and a first outlet tube, the end of the first injection tube extends into the first bottle, and the other part of the liquid to be separated that has not passed through the filter layer structure is discharged from the first bottle through the first outlet tube.

3. The centrifugal separation mechanism according to claim 1 or 2, characterized in that: The filter layer structure includes two membrane layers and a filling layer located between the two membrane layers. The pore size of the membrane layer is 6-10 μm, and the filling layer includes spherical particles with an outer diameter of 10-20 μm.

4. The centrifugal separation mechanism according to claim 3, characterized in that: The filter layer structure also includes a mesh support structure located outside the two membrane layers, and the mesh size of the support structure is larger than the pore size of the membrane layer.

5. The centrifugal separation mechanism according to claim 4, characterized in that: The material of the spherical particles includes polymethyl methacrylate; or / and, the spherical particles are composed of particles with an outer diameter of 10 μm and 20 μm mixed in equal proportions.

6. The centrifugal separation mechanism according to claim 4 or 5, characterized in that: The centrifugal separation mechanism also includes a collecting cover, which is covered on the side walls and bottom wall of the first bottle body and is used to collect the filtrate passing through the filter layer structure; preferably, the collecting cover is provided with a discharge port at a corresponding position of the bottom wall for draining out the filtrate.

7. A tumor cell separation device, characterized in that: It comprises an autologous blood flow path and a centrifugal separation mechanism as described in any one of claims 1 to 6; the centrifugal separation mechanism is arranged in the autologous blood flow path and is used to separate tumor cells in the autologous blood.

8. The tumor cell separation device according to claim 7, characterized in that: The tumor cell separation device also includes an inlet, a filter, a centrifugal cup and a blood storage device. The autologous blood flow path includes the inlet, the filter, the centrifugal separation mechanism, the centrifugal cup and the blood storage device connected in sequence; the filtrate of the centrifugal separation mechanism is stored in the blood storage device after passing through the centrifugal cup.

9. The tumor cell separation device according to claim 8, characterized in that: The centrifugal cup includes a second bottle body, which is configured to be a cone or a truncated cone with an axis vertically arranged and is rotatably arranged with the axis; the opening of the second bottle body is configured to face upward and is located on the end surface of the end with a smaller diameter; the opening of the second bottle body is connected to a second injection pipe and a second outlet pipe, the second injection pipe is connected to the interior of the second bottle body, and the filtrate is injected into the interior of the second bottle body; when the second bottle body is in a rotating state, the filtrate is centrifugally stratified, and the stratified filtrate is discharged respectively through the second outlet pipe.

10. The tumor cell separation device according to any one of claims 7 to 9, characterized in that: A driving pump is provided between the filter and the first bottle body, and between the first bottle body and the second bottle body, and the driving pump includes a peristaltic pump.

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