Portable device and method for peripheral blood circulating tumor cell clearance

Abstract

The invention provides a portable device and method for removing peripheral blood circulating tumor cells, and relates to the technical field of medical instruments, and the portable device mainly comprises a retention needle, a micro peristaltic pump, a flexible microfiltration membrane, a package of the flexible microfiltration membrane, a power supply and a silicone tube for connecting all the elements. According to the embodiment of the invention, the circulating tumor cells in the peripheral blood can be efficiently removed on the premise of no complex detection equipment, no label and no influence on the physiological state of the target animal.

Description

Technical Field

[0001] This invention relates to the field of medical device technology, and more particularly to a portable device and method for clearing tumor cells circulating in peripheral blood. Background Technology

[0002] Cancer is the second leading cause of death worldwide, and both incidence and mortality are increasing annually. Metastasis is a major factor contributing to cancer death, especially hematogenous metastasis. Circulating tumor cells (CTCs) play an irreplaceable role in hematogenous metastasis; preventing them from reaching distant organs through the bloodstream helps reduce the likelihood of cancer recurrence and metastasis.

[0003] To achieve the removal of CTCs from peripheral blood, the coupling method between the device and the blood vessel must first be determined. Currently, there are generally two methods: modifying the surface of a retention needle with specific antibodies / probes and inserting it directly into the blood vessel, and establishing an extracorporeal blood circulation system through cannulation surgery and connecting the device within it. The former is simple to operate and has a small device size, but it has drawbacks such as low blood throughput due to the blood flow rate in the blood vessel and a small effective area of ​​the sorting device due to the size of the blood vessel. The latter, although more complex to operate and with a larger sorting device, has advantages such as the ability to add an external driving source to increase blood throughput and compatibility with large-area sorting devices.

[0004] Secondly, it is necessary to determine the sorting method for CTCs. Currently, there are two main categories of CTC sorting methods: one based on biochemical properties (immunoaffinity), and the other based on physical properties (density, size, electrical properties, etc.). The former typically modifies the device surface with an antibody (e.g., anti-EpCAM antibody) corresponding to the CTC-specific antigen, and then couples the device to a blood vessel. When blood containing CTCs passes through the device, the CTCs are captured by the immunoaffinity, while background cells such as red blood cells and white blood cells can pass normally, thus achieving the sorting of CTCs in flowing blood. This type of method often has high specificity, but to ensure the efficiency of the immunoaffinity reaction, the throughput is often low, generally on the order of ~10 μL / min, limiting the volume of blood that can be processed. Furthermore, it may miss CTCs that do not express the target antigen.

[0005] Physical property-based methods generally rely on the significant differences in density, size, and electrical properties between central cytokines (CTCs) in peripheral blood and background cells (erythrocytes, leukocytes, etc.). Therefore, they can be separated from peripheral blood using methods such as density gradient centrifugation, filtration, fluid manipulation, and dielectrophoresis. These methods, to some extent, overcome the potential for missed detection of some CTCs by biochemical property-based methods, and do not require pre-labeling, ensuring cell viability and facilitating downstream analysis; theoretically, they also offer higher throughput. However, they suffer from limited specificity, meaning that some CTCs may overlap with background leukocytes or erythrocytes in physical properties, potentially leading to low sorting efficiency.

[0006] Finally, the captured CTCs must be removed from the system to achieve a "cleanup" effect. On one hand, for methods where the device is inserted into a blood vessel as an indwelling needle, external light or heat is typically used to inactivate the captured CTCs. This method may damage normal cells near the device, and the external auxiliary device is bulky and not suitable for long-term use. On the other hand, for methods that construct an extracorporeal circulation pathway, CTC removal can be achieved by replacing the device after a certain period of operation. Comparatively, the second method is more advantageous in reducing the overall size of the device, achieving a portable operation.

[0007] In summary, there is currently no new method that can efficiently remove all CTCs from peripheral blood. Summary of the Invention

[0008] The present invention provides a portable device and method for clearing circulating tumor cells in peripheral blood, thereby at least partially addressing the problems existing in the prior art.

[0009] The first aspect of the present invention provides a portable device for clearing tumor cells from peripheral blood circulation. The device includes: a first indwelling needle, a backpack, and a second indwelling needle; the first indwelling needle, the backpack, and the second indwelling needle are connected by a silicone tube, the first indwelling needle is located at the entrance side of the backpack, and the second indwelling needle is located at the exit side of the backpack. The backpack contains a flexible microporous filter membrane and its encapsulation, a micro-peristaltic pump, and a power supply. The micro-peristaltic pump, the flexible microporous filter membrane, and their encapsulation are connected by a silicone tube, and the micro-peristaltic pump and the power supply are connected by a wire.

[0010] Optionally, a PDMS three-way valve is further provided between the first retention needle and the backpack, and between the backpack and the second retention needle. The PDMS three-way valve includes a PDMS chip, a control circuit unit, a solenoid valve unit, and a power supply unit. The PDMS chip contains a liquid flow channel and a gas flow channel, and is externally connected through a capillary tube. The control circuit unit includes a microcontroller and a motor driver, and is connected to the micro peristaltic pump, the solenoid valve unit, and the PDMS chip to control the on / off state of the micro peristaltic pump and the six-way solenoid valve. Each solenoid valve in the solenoid valve unit is independently connected to nitrogen. When the solenoid valve is open, nitrogen is delivered to the gas flow channel in the corresponding PDMS chip, triggering valve diaphragm deformation and shutting off the liquid flow channel.

[0011] Optionally, the PDMS three-way valve is also connected to a sterile saline storage component.

[0012] Optionally, the backpack has two silicone tubes, six air ducts, and two wires leading out from the outside. The two silicone tubes are connected inside the backpack to the inlet of the PDMS chip liquid flow channel in the PDMS three-way valve on the inlet side and the outlet of the PDMS chip liquid flow channel in the PDMS three-way valve on the outlet side, respectively, and are connected to the first retention needle and the second retention needle on the outside. The air ducts are connected to the gas channels of the PDMS chip inside the backpack and to the solenoid valves in the solenoid valve unit outside the backpack. The wires connect the positive and negative terminals of the micro peristaltic pump inside the backpack, and connect the positive and negative terminals of the motor drive in the control circuit unit to the outside.

[0013] Optionally, the micro-peristaltic pump is connected to the microcontroller of the control circuit unit and the motor driver. The microcontroller outputs PWM waves with different duty cycles to the motor driver to adjust the flow rate of the micro-peristaltic pump.

[0014] Optionally, the encapsulation of the flexible microporous filter membrane includes a front-end encapsulation, a gasket, a rear-end encapsulation, and a fixing encapsulation. The front-end encapsulation and the rear-end encapsulation couple the silicone tube to the flexible microporous filter membrane. The gasket is used to prevent leakage. The fixing encapsulation detachably locks the front-end encapsulation and the rear-end encapsulation.

[0015] Optionally, the size of the flexible microporous filter membrane is determined based on the peripheral blood volume of the recipient, and the pore size of the flexible microporous filter membrane is determined based on the cell size of the recipient; the diameter and length of the silicone tube are determined based on the body size of the recipient; the size of the backpack is determined based on the size of the flexible microporous filter membrane and its encapsulation, the micro-peristaltic pump, and the power supply. The operating flow rate of the portable device is determined based on the recipient's adaptable in vitro blood circulation flow rate.

[0016] Optionally, the working time of the flexible microporous filter membrane is determined according to the following steps: Total blood volume based on the recipient V d Heart output Q h Determine the mixing time of the infinitesimal elements: ; Determine working hours T Post-circulating tumor cell concentration was C i : ; Based on the required reduction ratio of circulating tumor cell concentration in the blood of the recipient animal. , and preset workflow Q m Determine working hours T : .

[0017] A second aspect of this invention provides a method for clearing circulating tumor cells in peripheral blood, the method being applied to the portable device for clearing circulating tumor cells in peripheral blood described in the first aspect, the method comprising: Using the first indwelling needle as the input and the second indwelling needle as the output, fill all fluid channels with sterile saline. The PDMS three-way valve is used to connect the first indwelling needle, the micro-peristaltic pump, the flexible microporous filter membrane encapsulation back end, and the outlet waste liquid recovery branch. The inlet sterile saline branch and the second indwelling needle branch are disconnected. The first indwelling needle is connected to the jugular vein cannula of the recipient. The micro-peristaltic pump starts working at a preset flow rate. When the blood interface moves to a position close to the second indwelling needle, the outlet indwelling needle is connected to the carotid artery cannula of the recipient to begin filtering and removing circulating tumor cells in the peripheral blood. When the working time is reached or the flexible microporous filter membrane becomes severely clogged, the PDMS three-way valve is controlled to close the first indwelling needle and the second waste liquid recovery branch, and the sterile saline storage component, the micro peristaltic pump, the flexible microporous filter membrane encapsulation back end and the outlet indwelling needle branch are selected to open the micro peristaltic pump and return the peripheral blood remaining in the pipeline to the recipient device.

[0018] Optionally, the working time of the flexible microporous filter membrane is determined according to the following steps: Total blood volume based on the recipient V d Heart output Q h Determine the mixing time of the infinitesimal elements: ; Determine working hours T Post-circulating tumor cell concentration was C i : ; Based on the required reduction ratio of circulating tumor cell concentration in the blood of the recipient animal. , and preset workflow Q m Determine working hours T : .

[0019] The portable device provided in this invention mainly includes an indwelling needle, a micro-peristaltic pump, a flexible microporous filter membrane and its encapsulation, a power supply, and silicone tubing connecting the various components. This invention enables the efficient removal of circulating tumor cells from peripheral blood without complex detection equipment, labeling, or affecting the physiological state of the target animal.

[0020] The flexible microporous filter membrane used in this invention achieves CTC sorting based on differences in physical properties. It does not require complex detection equipment or labeling of target cells, and it does not introduce exogenous substances such as magnetic beads that may affect the treated blood. It can work without affecting the physiological state of the target animal.

[0021] The flexible microporous filter membrane used in this embodiment of the invention has the characteristics of precisely defined pore size, high porosity, and thin thickness, which ensures high capture rate and high activity of CTCs, low loss of background blood cells, and high throughput of the filtration process.

[0022] This invention integrates the core components, external piping, and driving peristaltic pump into a miniaturized backpack through integrated design, resulting in high portability and ensuring that the operation does not affect the basic behavior of the target object. Finally, this invention can set the working time of a single filter membrane by considering the capture efficiency of the flexible microporous filter membrane and the external circulation flow rate, and replace the device in a timely manner after the preset working time is reached, thus ensuring the effectiveness of CTC removal with minimal time cost. Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This diagram illustrates the structural composition of a portable device for clearing tumor cells from peripheral blood circulation, as provided in an embodiment of the present invention. Figure 2 This diagram illustrates the internal structure of a portable device for clearing tumor cells from peripheral blood circulation, as provided in an embodiment of the present invention. Figure 3 This invention provides a schematic diagram of the structure of a PDMS chip in a portable device for clearing tumor cells from peripheral blood circulation, as shown in an embodiment of the present invention. Figure 4 A schematic diagram of the structure of a backpack for a portable device for clearing peripheral blood circulating tumor cells provided in an embodiment of the present invention is shown; Figure 5 A schematic diagram of the structure of the flexible microporous filter membrane of the portable device for clearing peripheral blood tumor cells provided in an embodiment of the present invention is shown. Figure 6 This diagram illustrates the encapsulation structure of a flexible microporous filter membrane for a portable device suitable for clearing peripheral blood tumor cells in mice, as provided in an embodiment of the present invention. Figure 7 This diagram illustrates the encapsulation structure of a flexible microporous filter membrane for a portable device suitable for clearing peripheral blood tumor cells in rats, as provided in an embodiment of the present invention. Detailed Implementation

[0025] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0026] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents and other instruments whose manufacturers are not specified are all commercially available products.

[0027] This invention proposes to use a flexible microporous filter membrane for CTC sorting based on size differences as the core device. An extracorporeal circulation pathway is established through cannulation surgery, and the device is introduced into the blood circulation to capture CTCs. After working for a period of time, the core device is replaced to achieve long-term CTC clearance over time.

[0028] Specifically, firstly, the flexible microporous filter membrane used in this invention achieves CTC sorting based on differences in physical properties. It eliminates the need for complex detection equipment and target cell labeling, and avoids introducing exogenous substances such as magnetic beads that could affect the treated blood. It can operate without affecting the physiological state of the target animal. Secondly, the filter membrane itself features precisely definable pore size, high porosity, and thinness, ensuring a high CTC capture rate and high activity, low loss of background blood cells, and high throughput in the filtration process. Thirdly, this invention integrates the core components, external piping, and driving peristaltic pump into a miniaturized backpack, offering high portability and ensuring that the operation does not affect the basic behavior of the target animal. Finally, this invention allows for setting the working time of a single filter membrane based on the capture efficiency of the flexible microporous filter membrane and the in vitro circulation flow rate, and promptly replaces the device after the preset working time, ensuring the effectiveness of CTC removal with minimal time cost.

[0029] Specifically, Figure 1 A schematic diagram of the structure of a portable device for clearing tumor cells in peripheral blood circulation provided by an embodiment of the present invention is shown. The device includes: a first indwelling needle, a backpack, and a second indwelling needle; the first indwelling needle, the backpack, and the second indwelling needle are connected by a silicone tube, the first indwelling needle is located on the inlet side of the backpack, and the second indwelling needle is located on the outlet side of the backpack. The backpack contains a flexible microporous filter membrane and its encapsulation, a micro-peristaltic pump, and a power supply. The micro-peristaltic pump, the flexible microporous filter membrane, and their encapsulation are connected by a silicone tube, and the micro-peristaltic pump and the power supply are connected by a wire.

[0030] In this embodiment of the invention, the indwelling needles at the inlet and outlet serve to establish an extracorporeal blood circulation system for the recipient without affecting normal physiological state and life activities. Both indwelling needles and the backpack are connected by silicone tubing. The miniaturized backpack is the core hardware device of this embodiment, and its function is to capture and remove central thrombi (CTCs) in the extracorporeal blood circulation pathway in real time to achieve a therapeutic effect.

[0031] The miniaturized backpack contains a micro-peristaltic pump, a flexible microporous filter membrane and its encapsulation, and a power supply. The micro-peristaltic pump applies external force to maintain extracorporeal blood circulation. The flexible microporous filter membrane is the core component of this invention. Through precise pore size design, it allows small background leukocytes and erythrocytes in peripheral blood to pass through normally, while larger CTCs are intercepted and captured. Furthermore, by replacing the flexible microporous filter membrane after a period of operation, the CTCs captured on the previous filter membrane can be removed, and the ability to capture CTCs can be resumed. The flexible microporous filter membrane is fixed by a 3D-printed encapsulation. The peristaltic pump and the flexible microporous filter membrane inside the backpack are also connected by a silicone tube, which is connected to an external retention needle. The power supply provides power to the micro-peristaltic pump. The flow rate of the peristaltic pump in the basic architecture does not need to be adjusted, so only DC power is required.

[0032] In this embodiment of the invention, different models of peristaltic pumps may be used, and the flow rate range and volume may vary, but all can achieve the same effect.

[0033] The portable device for clearing circulating tumor cells in peripheral blood provided in this invention can achieve in vivo clearance of CTCs. During operation, it does not have a significant negative impact on background leukocytes and erythrocytes, has a high CTC capture efficiency, and there is no risk of re-infusing captured CTCs into the animal during the filtration process.

[0034] Furthermore, the portable device for clearing tumor cells in peripheral blood circulation provided in this embodiment of the invention does not impose a significant burden on the recipient and does not affect their normal life activities.

[0035] This invention also provides a schematic diagram of a portable device for clearing tumor cells from peripheral blood circulation, as shown in the embodiments of the invention. Figure 2 As shown, specifically, a PDMS (polydimethylsiloxane) three-way valve is also provided between the first retention needle and the backpack, and a PDMS three-way valve is also provided between the backpack and the second retention needle. The PDMS three-way valve includes: a PDMS chip, a control circuit unit, a solenoid valve unit, and a power supply unit. The PDMS chip contains a liquid flow channel, a gas flow channel, and a glass substrate, and is externally connected through a capillary tube. The control circuit unit includes a microcontroller and a motor driver, and is connected to the micro peristaltic pump, the solenoid valve unit, and the PDMS chip to control the on / off state of the micro peristaltic pump and the six solenoid valves. Each solenoid valve in the solenoid valve unit is independently connected to nitrogen. When the solenoid valve is open, nitrogen is delivered to the gas flow channel in the corresponding PDMS chip, triggering valve diaphragm deformation and shutting off the liquid flow channel.

[0036] In this embodiment of the invention, the PDMS three-way valve is also connected to a sterile saline storage component. Figure 2 The device comprises, from front to back, a first indwelling needle (inlet), sterile saline solution, a PDMS chip, a micro-peristaltic pump, a flexible microporous filter membrane and its encapsulation, and a second indwelling needle (outlet). The components are connected by silicone tubes.

[0037] The indwelling needle can employ any feasible technology from relevant fields, primarily consisting of a needle tip and a fixation structure. It establishes an extracorporeal blood circulation pathway by puncturing a vein / artery and temporarily fixing it in place. The sterile saline solution serves to cleanse any residual blood inside the device, preventing blood from remaining in the silicone tube for extended periods and causing clotting during the replacement of the flexible microporous filter membrane. It can be packaged in either bottles or bags.

[0038] Figure 3 This diagram illustrates the structure of the PDMS chip in a portable device for clearing tumor cells from peripheral blood circulation, provided in an embodiment of the present invention. The PDMS chip controls the opening and closing of the extracorporeal blood circulation pathway, the saline washing pathway, and the waste liquid pathway. To further reduce the device size and minimize the dead volume inside the valves, this embodiment uses two sets of PDMS three-way valves. Each PDMS valve specifically includes a PDMS chip, a control circuit unit, a solenoid valve unit, and a power supply unit.

[0039] like Figure 3 As shown, the PDMS chip contains fluid channels and gas channels, and is externally connected via capillary tubes. The specific processing flow is as follows: First, a fluid channel mold is fabricated using photoresist (such as AZ4620), with a thickness of approximately 30 μm, and then reflowed. After reflow, the channel cross-section is semi-circular, making it easy to be cut off by the gas channel. Second, a gas channel mold is fabricated using photoresist (such as SU82015), with a thickness of approximately 20 μm. Third, PDMS with a 1 / 10 ratio is spin-coated onto the surface of the gas channel mold, with a thickness of approximately 30 μm, to obtain the gas channel PDMS channel and valve membrane. At this point, the valve membrane thickness is ~10 μm. Fourth, the fluid channel mold is cast using PDMS with a 1 / 10 ratio. After curing, a liquid channel PDMS channel is formed, which is then bonded to the gas channel PDMS channel obtained in the third step through oxygen plasma treatment. Fifth, the PDMS chip obtained in the fourth step is peeled off from the gas channel mold and bonded to a glass substrate after oxygen plasma treatment.

[0040] The control circuit unit mainly consists of a microcontroller and a motor driver, connected to a miniature peristaltic pump, a solenoid valve unit, and two sets of PDMS three-way valves. Its function is to control the on / off state of the peristaltic pump and the six-way solenoid valves as needed. Each solenoid valve in the solenoid valve unit is independently connected to nitrogen gas. Preferably, the nitrogen pressure is 0.2~0.5MPa. The control unit controls the valve opening and closing. When the valve opens, nitrogen gas is delivered to the gas channel within the corresponding PDMS chip, triggering valve diaphragm deformation and shutting off the liquid flow. The power supply unit includes a battery and a step-down module. The battery voltage can be 12-24V, directly powering the solenoid valve unit. Simultaneously, the step-down module adjusts the voltage to 5V or 3.7V to further power the control circuit unit, the motor driver, and the miniature peristaltic pump.

[0041] The PDMS chip is located inside the backpack, while the control circuit unit, solenoid valve unit, and power supply unit are located outside the backpack.

[0042] In this embodiment of the invention, a 3D-printed "backpack" was designed, as shown in the attached figure. Figure 4 As shown, the aforementioned PDMS three-way valve, micro peristaltic pump, flexible microporous filter membrane, and their packaging fixture are uniformly encapsulated within a single unit, with internal connections similar to... Figure 2 The backpack dimensions are determined based on actual needs. Two silicone tubes, six air ducts, and two wires extend from the outside of the backpack. The two silicone tubes connect inside the backpack to the inlet and outlet of the PDMS chip liquid flow channel in the PDMS three-way valve, respectively, and externally to the first and second retention needles, respectively. The air ducts connect inside the backpack to the gas flow channels of the PDMS chip, and externally to the solenoid valves in the solenoid valve unit. The wires connect inside the backpack to the positive and negative terminals of the micro peristaltic pump, and externally to the positive and negative terminals of the motor drive in the control circuit unit, respectively.

[0043] In this embodiment of the invention, the backpack can be made of resin and processed by 3D printing technology. It can also be processed by different methods such as machining and injection molding. As long as there is a similar structure, the same effect can be achieved.

[0044] In this embodiment of the invention, the micro-peristaltic pump is connected to a microcontroller in the control circuit unit and a motor driver. The microcontroller outputs PWM waves with different duty cycles to the motor driver to adjust the flow rate of the micro-peristaltic pump. The adjustment range is generally 10%-100%, allowing the peristaltic pump flow rate to be adjusted within the range of 0.03 mL / min to 0.1 mL / min. In this embodiment of the invention, different models of the micro-peristaltic pump can be selected according to specific application scenarios to achieve different flow rates.

[0045] The flexible microporous filter membrane is the core component of this invention, such as... Figure 5As shown, it features precisely defined pore size, high porosity, and ultra-thin thickness, and is manufactured using microelectromechanical systems (MEMS) technology.

[0046] The specific processing dimensions can be determined based on the cell size and body size of the recipient. Taking mice as an example, the specific processing flow is as follows: First, a micropillar array mask is formed on the silicon substrate using a thin photoresist. The micropillars are hexagonal in shape, with an outer circle diameter of 8-10 μm and a spacing of 4 μm. Second, deep silicon etching is performed to a depth of 10-12 μm. After etching, the photoresist mask is removed, forming a silicon micropillar array. Third, Parylene C material with a thickness of ~2 μm is deposited on the substrate surface. Fourth, the membrane is immersed in an HNA solution prepared using hydrofluoric acid, nitric acid, and acetic acid in a volume ratio of 40 / 70 / 110. Immersion releases the Parylene C flexible microporous filter membrane from the silicon substrate surface. Fifth, the flexible microporous filter membrane is cut to obtain a square shape with a side length of 5 mm. Based on the above processing flow, the etching mask is made of thin photoresist and photolithography, which can achieve precise pore size definition and high porosity; while the thickness of the Parylene C microporous filter membrane is determined by the height of the silicon micropillar array, which is generally around 10μm, thus it has the characteristic of ultra-thin thickness.

[0047] In this embodiment of the invention, the flexible microporous filter membrane is made of Parylene C. Flexible microporous filter membranes made of different materials, such as SU 8, PDMS, or silicon, can achieve the same effect as long as the pore size, pore spacing, and thickness are the same as in this embodiment.

[0048] To couple the flexible microporous filter membrane with other components, this embodiment also includes a corresponding encapsulation device, as shown in the attached diagram. Figure 6 As shown. The encapsulation of the flexible microporous filter membrane includes a front-end encapsulation, a gasket, a rear-end encapsulation, and a fixing encapsulation. The front-end and rear-end encapsulations couple the silicone tube to the flexible microporous filter membrane. The gasket is used to prevent leakage. The fixing encapsulation detachably locks the front-end and rear-end encapsulations.

[0049] In this embodiment of the invention, the key consideration in the packaging design of the flexible microporous filter membrane is its filtration area and volume. Theoretically, the larger the filtration area, the larger the volume of blood that can be processed. However, an excessively large packaging volume can lead to an excessively large blood volume in the animal's extracorporeal blood circulation pathway, affecting normal life activities. Therefore, the packaging volume of the aforementioned flexible microporous filter membrane is ~0.1mL, specifically including front-end packaging, gasket, rear-end packaging, and fixing packaging. The front-end packaging couples the silicone tube to the flexible microporous filter membrane, with the inlet cross-section having a critical dimension of 0.6mm inner diameter. The gasket prevents leakage, preferably with an outer diameter of 0.5mm and an inner diameter of 0.3mm. The rear-end packaging has the same function as the front-end packaging, also coupling the flexible microporous filter membrane to the silicone tube; therefore, the outlet cross-section also has a critical dimension of 0.6mm inner diameter. The fixing packaging securely locks the front-end and rear-end packaging, and the gasket helps prevent leakage during operation. The outlet retention needle is identical to the inlet retention needle and will not be elaborated here.

[0050] In this embodiment of the invention, the flexible microporous filter membrane encapsulation material can be any feasible material, such as PMMA.

[0051] In this embodiment of the invention, the size of the flexible microporous filter membrane is determined based on the peripheral blood volume of the recipient, and the pore size of the flexible microporous filter membrane is determined based on the cell size of the recipient; the diameter and length of the silicone tube are determined based on the body size of the recipient; and the size of the backpack is determined based on the size of the flexible microporous filter membrane and its encapsulation, the micro-peristaltic pump, and the power supply.

[0052] The operating flow rate of the portable device is determined based on the recipient's adaptable in vitro blood circulation flow rate.

[0053] In this embodiment of the invention, the inner diameter of the silicone tube is determined based on the peripheral blood volume of the target animal. An inner diameter that is too small will result in low flow rate in the extracorporeal blood circulation pathway, making thrombus formation easier and leading to a low CTC purification rate; an inner diameter that is too large will result in an excessively large blood volume in the extracorporeal blood circulation pathway, affecting the physiological state of the target animal. It is generally considered that a safe blood volume in the extracorporeal blood circulation pathway is 10%-20% of the total blood volume. Taking mice as an example, the peripheral blood volume of mice is generally considered to be around 1 mL, so the total blood volume in the extracorporeal blood circulation pathway does not exceed 0.2 mL. Considering that the volume of the aforementioned flexible microporous filter membrane encapsulation is 0.1 mL, the silicone tube is set with an inner diameter of 0.5 mm and an outer diameter of 0.6 mm, with a total length of ~200 mm, and the estimated occupied volume is 0.04 mL. Furthermore, the PDMS valve chip channel height is ~30 μm, and the occupied volume is negligible. Therefore, under the combined effect, the blood volume in the extracorporeal blood circulation pathway is ~0.14 mL, which theoretically does not affect the normal physiological activities of the mouse.

[0054] The size design of each component of the portable device provided in this invention can be specifically designed for the blood volume in the extracorporeal blood circulation pathway of the recipient, and can be applied to any type of recipient, such as mice, rats, pit bulls, humans, etc.

[0055] The size design of each component of the portable device provided in this embodiment of the invention takes into account the body size of the recipient, and does not affect the normal physiological activities of the recipient during the filtering of circulating tumor cells, making it suitable for clinical treatment.

[0056] The portable device provided in this embodiment of the invention is mainly used for the removal of circulating tumor cells in peripheral blood. Theoretically, the longer the working time, the better the removal effect. However, in practice, excessively long working times may cause the circulating tumor cells captured by the flexible microporous filter membrane to deform under prolonged fluid impact and be reinfused into the animal. Therefore, it is necessary to determine an appropriate working time. Due to the function of the animal's heart, the blood present in extracorporeal circulation at each time period is the same as that in the previous time period. Therefore, it cannot be calculated using a simple volume / flow rate method. This embodiment of the invention establishes a first-order kinetic model for estimation.

[0057] Specifically, the working time of the flexible microporous filter membrane is determined according to the following steps: Total blood volume based on the recipient V d Heart output Q h Determine the mixing time of the infinitesimal elements: ; Determine working hours T Post-circulating tumor cell concentration was C i : ; Based on the required reduction ratio of circulating tumor cell concentration in the blood of the recipient animal. , and preset workflow Q m Determine working hours T : .

[0058] In this embodiment of the invention, the specific time the device needs to operate can be determined according to the above formula. For example, when the total blood volume of the target animal is... V d =1mL, cardiac output is Q h =1 mL / min, initial concentration of circulating tumor cells was C 0, extracorporeal flow rate is Qm With a flow rate of 0.1 mL / min, a capture rate of P=85%, and a working time of T=30 min, the percentage of circulating tumor cells reduced after working was ~93.0%. Therefore, to achieve the removal of ~90% of circulating tumor cells in peripheral blood, a working time of 30 min is required.

[0059] In this embodiment of the invention, the portable device described above can be used to separate circulating tumor cells from the peripheral blood of live animals. The working time required to achieve effective removal can be determined based on the formula described above. By separating circulating tumor cells from the peripheral blood of the recipient based on this working time, therapeutic applications can be achieved.

[0060] In this embodiment of the invention, the core device for CTC capture is based on a physical property sorting principle, which differs from a biochemical property sorting principle. This avoids complex and bulky detection and sorting equipment, eliminates the need to label target cells, and avoids introducing exogenous substances such as magnetic beads that could affect the treated background leukocytes and erythrocytes. This achieves efficient removal of CTCs from peripheral blood without affecting the physiological state of the target animal.

[0061] In this embodiment of the invention, the core device is a flexible microporous filter membrane, which has the characteristics of precisely defined pore size, high porosity, and thin thickness, ensuring high capture filtration in the CTC capture process and achieving efficient removal of CTCs from peripheral blood.

[0062] The embodiments of the present invention integrate the core components, external piping, and driving peristaltic pump into a "miniaturized backpack" through integrated design, which is highly portable and does not affect the behavior of the target object during operation.

[0063] Finally, the embodiments of the present invention can estimate the working time of a single filter membrane based on the extracorporeal circulation flow rate, and replace the device in a timely manner after the preset working time is reached, thus ensuring the effectiveness of CTC removal in peripheral blood over time. It can achieve in vivo clearance of CTC over a long period of time. The device can be replaced in a timely manner after the CTC capture capacity is saturated, ensuring that a high CTC binding capacity is maintained. Moreover, the device replacement time can be determined by a kinetic model.

[0064] This invention also provides a non-therapeutic method for the removal of circulating tumor cells in peripheral blood, wherein the method is applied to the portable device for the removal of circulating tumor cells in peripheral blood described in any of the above embodiments, and the method includes: S101 uses the first indwelling needle as input and the second indwelling needle as output, filling all fluid channels with sterile saline.

[0065] S102, the PDMS three-way valve is used to select the first indwelling needle, the micro-peristaltic pump, the flexible microporous filter membrane encapsulation back end, and the outlet waste liquid recovery branch. The inlet sterile saline branch and the second indwelling needle branch are disconnected. The first indwelling needle is connected to the jugular vein cannula of the recipient. The micro-peristaltic pump starts working at a preset flow rate. When the blood interface moves to a position close to the second indwelling needle, the outlet indwelling needle is connected to the carotid artery cannula of the recipient to begin filtering and removing circulating tumor cells in the peripheral blood.

[0066] S103, when the working time is reached or the flexible microporous filter membrane is severely blocked, controls the PDMS three-way valve to close the first indwelling needle and the second waste liquid recovery branch, selects the sterile saline storage component, the micro peristaltic pump, the flexible microporous filter membrane encapsulation back end and the outlet indwelling needle branch, and opens the micro peristaltic pump to return the peripheral blood stored in the pipeline to the recipient device.

[0067] Specifically, taking mice as the recipient, the experimental procedure is as follows: First, sterilization: the flexible microporous filter membrane and PDMS three-way valve / PDMS chip were soaked in 75% alcohol for 15 minutes, and other materials that come into contact with blood were also soaked in 75% alcohol for 15 minutes, followed by sterilization using a standard biosafety cabinet with UV irradiation for 30 minutes. Second, installation and pretreatment: all materials were connected using silicone tubing or wires. After connection, the micro-peristaltic pump and PDMS three-way valve were tested. Next, using the inlet indwelling needle as input and the outlet indwelling needle as output, all fluid channels were filled with sterile physiological saline. The third step is the filtration operation. The PDMS three-way valve is used to open the inlet indwelling needle, the micro-peristaltic pump, the flexible microporous membrane encapsulation back end, and the outlet waste liquid recovery branch. The inlet sterile saline branch and the outlet indwelling needle branch are disconnected. The inlet indwelling needle is connected to the mouse jugular vein cannula. The micro-peristaltic pump starts operating at a flow rate of 0.03-0.1 mL / min. When the blood interface moves close to the outlet indwelling needle, the outlet indwelling needle is connected to the mouse carotid artery cannula to begin filtration and remove circulating tumor cells from the peripheral blood. The fourth step is to terminate filtration and replace the flexible microporous membrane. When the filtration time is reached or the flexible microporous membrane becomes severely clogged, the three-way valve is used to close the inlet indwelling needle and the outlet waste liquid recovery branch. The sterile saline, micro-peristaltic pump, flexible microporous membrane encapsulation back end, and outlet indwelling needle branch are then opened. The micro-peristaltic pump is turned on for approximately 1 minute to ensure that most of the peripheral blood remaining in the tubing is reinfused into the mouse, minimizing blood loss. Disconnect the inlet and outlet retaining needles, remove the used flexible microporous filter membrane, and replace it with a new one.

[0068] Specifically, taking rats as the recipient, the overall hardware device is similar to the above embodiments, only the flexible microporous filter membrane and its encapsulation, the silicone tube, and the "backpack" are replaced, such as... Figure 7As shown in sections (a) and (b). Specifically, considering that the peripheral blood volume of rats is ~10mL, and to ensure the normal functioning of rats, the peripheral blood volume in the extracorporeal blood circulation pathway is 1mL-2mL, therefore the encapsulation volume of the flexible microporous filter membrane is ~1mL. The pore size of the flexible microporous filter membrane can be selected as 8-10μm, the pore spacing is 4μm, and the processing flow is the same as the aforementioned method, but the final step of cutting the shape is changed to a square with a side length of 10mm. The encapsulation structure of the flexible microporous filter membrane is also similar to the aforementioned method, only the characteristic dimensions are changed: the key dimension of the front encapsulation inlet is a circle with a cross-sectional diameter of 7mm and a length of ~17mm, and the key dimension of the rear encapsulation outlet is a circle with a cross-sectional diameter of 7mm and a length of ~17mm. At this time, the overall internal volume of the encapsulation is ~1.3mL. In this encapsulation, compared with Example 1, the effective filtration area of ​​the flexible microporous filter membrane is increased. To avoid damage to the filter membrane during filtration, a cross-shaped support structure is also set in the rear encapsulation. The thickness and width of the support structure are both 1mm, and the length is 7mm, as shown. Figure 3 As shown in section (b).

[0069] Accordingly, the size of the silicone tube was also adjusted, changing it to an outer diameter of 3mm and an inner diameter of 1mm, with the length also limited to ~200mm. At this point, the blood volume in the silicone tube portion is 0.1mL. After the device is integrated, the total blood volume is ~1.4mL, which theoretically does not affect the physiological state and vital activities of the rat. In addition, to accommodate the larger volume of the flexible microporous filter membrane, the size of the "backpack" was also increased accordingly, but the basic structure is the same as in the aforementioned embodiment, and will not be described in detail here.

[0070] The experimental procedure was similar to that described above. Considering that rats can be compatible with higher flow rates in vitro blood circulation, the working flow rate was set at 1-2 mL / min to improve purification efficiency.

[0071] In this embodiment of the invention, the method for clearing tumor cells in the peripheral blood circulation is a non-therapeutic, non-diagnostic method, such as a research method for model animals.

[0072] Although preferred embodiments of the present invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the embodiments of the present invention.

[0073] The above provides a detailed description of a portable device and method for clearing tumor cells in peripheral blood circulation provided by the present invention. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A portable device for clearing tumor cells from peripheral blood circulation, characterized in that, The device includes: a first retention needle, a backpack, and a second retention needle; the first retention needle, the backpack, and the second retention needle are connected by a silicone tube, the first retention needle is located on the inlet side of the backpack, and the second retention needle is located on the outlet side of the backpack; The backpack contains a flexible microporous filter membrane and its encapsulation, a micro-peristaltic pump, and a power supply. The micro-peristaltic pump, the flexible microporous filter membrane, and their encapsulation are connected by a silicone tube, and the micro-peristaltic pump and the power supply are connected by a wire.

2. The portable device for clearing tumor cells from peripheral blood circulation according to claim 1, characterized in that, A PDMS three-way valve is also provided between the first retention needle and the backpack, and between the backpack and the second retention needle. The PDMS three-way valve includes a PDMS chip, a control circuit unit, a solenoid valve unit, and a power supply unit. The PDMS chip contains a liquid flow channel, a gas flow channel, and a glass substrate, and is externally connected through a capillary tube. The control circuit unit includes a microcontroller and a motor driver, and is connected to the micro peristaltic pump, the solenoid valve unit, and the PDMS chip to control the on / off state of the micro peristaltic pump and the six solenoid valves. Each solenoid valve in the solenoid valve unit is independently connected to nitrogen. When the solenoid valve is open, nitrogen is delivered to the gas flow channel in the corresponding PDMS chip, triggering valve diaphragm deformation and shutting off the liquid flow channel.

3. The portable device for clearing tumor cells from peripheral blood circulation according to claim 2, characterized in that, The PDMS three-way valve is also connected to a sterile saline storage component.

4. The portable device for clearing tumor cells from peripheral blood circulation according to claim 3, characterized in that, The backpack has two silicone tubes, six air ducts, and two wires leading out from the outside. The two silicone tubes are connected inside the backpack to the inlet of the PDMS chip liquid flow channel in the PDMS three-way valve on the inlet side and the outlet of the PDMS chip liquid flow channel in the PDMS three-way valve on the outlet side, respectively. They are connected to the first retention needle and the second retention needle on the outside. The air ducts are connected to the gas channels of the PDMS chip inside the backpack and to the solenoid valves in the solenoid valve unit outside the backpack. The wires connect the positive and negative terminals of the micro peristaltic pump inside the backpack, and connect the positive and negative terminals of the motor drive in the control circuit unit to the outside.

5. The portable device for clearing tumor cells from peripheral blood circulation according to claim 2, characterized in that, The micro-peristaltic pump is connected to the microcontroller of the control circuit unit and the motor driver. The microcontroller outputs PWM waves with different duty cycles to the motor driver to adjust the flow rate of the micro-peristaltic pump.

6. The portable device for clearing tumor cells from peripheral blood circulation according to claim 1, characterized in that, The encapsulation of the flexible microporous filter membrane includes a front-end encapsulation, a gasket, a rear-end encapsulation, and a fixing encapsulation. The front-end encapsulation and the rear-end encapsulation couple the silicone tube to the flexible microporous filter membrane. The gasket is used to prevent leakage. The fixing encapsulation detachably locks the front-end encapsulation and the rear-end encapsulation.

7. The portable device for clearing tumor cells from peripheral blood circulation according to claim 1, characterized in that, The size of the flexible microporous filter membrane is determined based on the peripheral blood volume of the recipient, and the pore size of the flexible microporous filter membrane is determined based on the cell size of the recipient; the diameter and length of the silicone tube are determined based on the body size of the recipient; the size of the backpack is determined based on the size of the flexible microporous filter membrane and its encapsulation, the micro-peristaltic pump, and the power supply. The operating flow rate of the portable device is determined based on the recipient's adaptable in vitro blood circulation flow rate.

8. The portable device for clearing tumor cells from peripheral blood circulation according to claim 1, characterized in that, The working time of the flexible microporous filter membrane is determined according to the following steps: Total blood volume based on the recipient V d Heart output Q h Determine the mixing time of the infinitesimal elements: ； Determine working hours T Post-circulating tumor cell concentration was C i : ； Based on the required reduction ratio of circulating tumor cell concentration in the blood of the recipient animal. , and preset workflow Q m Determine working hours T : 。 9. A non-therapeutic method for the removal of circulating tumor cells in peripheral blood, characterized in that, The method is applied to the portable device for clearing peripheral blood circulating tumor cells according to any one of claims 1-8, the method comprising: Using the first indwelling needle as the input and the second indwelling needle as the output, fill all fluid channels with sterile saline. The PDMS three-way valve is used to connect the first indwelling needle, the micro-peristaltic pump, the flexible microporous filter membrane encapsulation back end, and the outlet waste liquid recovery branch. The inlet sterile saline branch and the second indwelling needle branch are disconnected. The first indwelling needle is connected to the jugular vein cannula of the recipient. The micro-peristaltic pump starts working at a preset flow rate. When the blood interface moves to a position close to the second indwelling needle, the outlet indwelling needle is connected to the carotid artery cannula of the recipient to begin filtering and removing circulating tumor cells in the peripheral blood. When the working time is reached, the PDMS three-way valve closes the first indwelling needle and the second waste liquid recovery branch, selects the sterile saline storage component, the micro peristaltic pump, the flexible microporous filter membrane encapsulation back end and the outlet indwelling needle branch, and opens the micro peristaltic pump to return the peripheral blood stored in the pipeline to the recipient device.

10. The method for clearing circulating tumor cells in peripheral blood according to claim 9, characterized in that, The working time of the flexible microporous filter membrane is determined according to the following steps: Total blood volume based on the recipient V d Heart output Q h Determine the mixing time of the infinitesimal elements: ； Determine working hours T Post-circulating tumor cell concentration was C i : ； Based on the required reduction ratio of circulating tumor cell concentration in the blood of the recipient animal. , and preset workflow Q m Determine working hours T : 。

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