A highly efficient continuous extraction system for vesicles from human umbilical cord mesenchymal stem cells

Abstract

This invention relates to the fields of bioengineering and medical device technology, specifically to a highly efficient continuous extraction system for vesicles from human umbilical cord mesenchymal stem cells. The system includes an ultrasonic unit, a centrifugation unit, a size exclusion chromatography column purification unit, a collection unit, a closed-loop tubing system, and a control unit. This highly efficient continuous extraction system for human umbilical cord mesenchymal stem cells integrates three key technology modules—ultrasound, centrifugation, and chromatographic purification—and automates and closes the process, significantly reducing manual operation and sample exposure time, effectively lowering the risk of contamination, and improving extraction efficiency, purity, and activity of the vesicle products.

Description

Technical Field

[0001] This invention relates to the fields of bioengineering and medical device technology, specifically to a highly efficient continuous extraction system for vesicles from human umbilical cord mesenchymal stem cells. Background Technology

[0002] Intracellular vesicles, including exosomes and microvesicles, are important mediators of intercellular communication, carrying bioactive substances such as proteins, nucleic acids, and lipids, and show great potential in tissue repair, immune regulation, and disease treatment. Vesicles derived from umbilical cord mesenchymal stem cells have become a research hotspot in regenerative medicine due to their low immunogenicity and multiple biological functions.

[0003] Currently, mainstream methods for extracting intracellular vesicles typically involve a combination of multiple independent steps. For example, cells may be treated with sonication, freeze-thaw cycles, or chemical reagents to release vesicles, followed by ultracentrifugation to remove cell debris, and finally purification using ultrafiltration, precipitation, or size exclusion chromatography. These methods have significant limitations: multiple steps require manual sample transfer between different devices, which is cumbersome, time-consuming, and prone to introducing contamination or vesicle loss; effective temperature control throughout the process is lacking, especially since sonication can generate heat, potentially damaging vesicle integrity and bioactivity; while traditional ultracentrifugation is commonly used, it can damage vesicle structures due to high shear forces, and the equipment is expensive with limited throughput.

[0004] While existing technologies disclose devices for centrifuging cell vesicles, their functions are limited, focusing solely on the centrifugation process and lacking integration with upstream cell disruption and downstream fine purification techniques. Therefore, developing an integrated extraction instrument capable of achieving a fully automated, temperature-controlled process from cell processing to high-purity vesicle acquisition is crucial for promoting the standardized preparation and clinical application of intracellular vesicles. Summary of the Invention The purpose of this invention is to overcome the shortcomings of existing technologies, such as multi-step separation, cumbersome operation, low efficiency, and large loss of activity, and to provide a highly efficient and continuous extraction system for intracellular vesicles of human umbilical cord mesenchymal stem cells. This system integrates key extraction steps into a closed and coherent system, and the operation process is automatically and continuously carried out from ultrasound, aiming to extract intracellular vesicles of umbilical cord mesenchymal stem cells efficiently, with high purity and high activity.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A highly efficient continuous extraction system for vesicles from human umbilical cord mesenchymal stem cells, comprising: An ultrasound unit is used to break up cells to release intracellular vesicles. The ultrasound unit is equipped with a sample processing chamber with a water-proof function, and the bottom of the sample processing chamber is equipped with a liquid outlet.

[0006] The centrifugal separation unit includes a centrifuge chamber, a rotatable centrifugal rotor, a liquid inlet module, a supernatant outlet module, and a replacement door located on the side wall of the centrifuge chamber. The centrifugal rotor has at least one centrifuge bottle with its opening facing upwards. The liquid inlet module is connected to the liquid outlet of the processing chamber and has a liftable liquid inlet needle. The supernatant outlet module has a supernatant outlet and a liftable liquid suction needle. The liquid inlet needle, liquid suction needle, and centrifugal rotor are all located inside the centrifuge chamber. The centrifugal rotor can rotate and position the centrifuge bottle directly below the liquid inlet needle or the liquid suction needle, or at the replacement door.

[0007] The size exclusion chromatography column purification unit is connected to the supernatant outlet of the centrifugation separation unit and is used for separation and enrichment based on vesicle particle size.

[0008] The collection unit is connected to the size exclusion chromatography column purification unit to collect the final product.

[0009] A closed-loop piping system, including the connections between the above-mentioned units and the provision of liquid transport power, is used for the continuous flow of samples in a closed environment.

[0010] The control unit, connected to all the above units, is used to control the entire process operation.

[0011] Furthermore, the ultrasonic unit is equipped with an ultrasonic generator that applies ultrasonic waves to the sample processing chamber. The outer layer of the sample processing chamber is equipped with a jacket structure for circulating coolant. The jacket structure is connected to a circulating cooling system. A temperature sensor is installed inside the jacket to ensure that the ultrasonic process is carried out at a low temperature.

[0012] Furthermore, the closed-loop piping system includes a smooth-walled medical-grade silicone tube connected to the liquid outlet of the treatment chamber. The medical-grade silicone tube passes through an electric clamp valve or a solenoid valve and is then connected to the liquid inlet module of the centrifugal separation unit.

[0013] Furthermore, the inlet needle and the suction needle can be raised and lowered by any one of the following structures: an electric push rod, a lead screw assembly, or a gear and rack assembly.

[0014] Furthermore, the centrifuge bottle is sealed with a self-sealing rubber diaphragm.

[0015] Furthermore, the lifting mechanism of the aspiration needle is provided with an upper limit position and a lower limit position. The lower limit position is adjusted to a set distance above the top surface of the theoretical sediment layer at the bottom of the centrifuge bottle to ensure that the aspiration needle will not enter the sediment layer.

[0016] Furthermore, a concave structure is provided at the center of the bottom of the centrifuge bottle. The height of the concave structure is slightly higher than the height of the precipitate. When the aspiration needle descends to a certain extent, it will encounter the concave structure, at which point the aspiration needle can no longer move down.

[0017] Furthermore, the aspiration needle is covered with an insulating layer to form a capacitor, with the needle body as one pole and the liquid it contacts as the other pole. When the tip of the aspiration needle contacts the liquid surface, the capacitance value changes abruptly. The controller immediately records the current position pulse count and stops descending. Then, a descent depth is set from the stop position to begin aspiration. During the aspiration process, the capacitance change is monitored in real time. If the capacitance value rebounds, the controller sends a pulse to the stepper motor to lower the aspiration needle, ensuring that the needle tip is always immersed in the supernatant.

[0018] Furthermore, the size exclusion chromatography column of the purification unit is made of glass or biocompatible polymer, and the two ends of the column are equipped with replaceable sieve plates and mobile phase distributors.

[0019] Furthermore, the inner diameter of the size exclusion chromatography column ranges from 15 to 40 mm, the length ranges from 100 to 200 mm, and the packing material inside the column is a size exclusion medium based on agarose or dextran, with an exclusion limit of 35 nm and a separation range of 30 to 200 nm.

[0020] The present invention has the following advantages: This invention discloses a highly efficient and continuous extraction system for intracellular vesicles from human umbilical cord mesenchymal stem cells. By organically integrating three core technology modules—ultrasonic disruption, centrifugation, and chromatographic purification—a fully enclosed, automated, and continuous intracellular vesicle extraction system is constructed. This system completely changes the traditional multi-step, manual, and fragmented equipment approach, achieving full automation from cell disruption to high-purity vesicle collection through precise timing control and parameter optimization.

[0021] This invention provides a highly efficient continuous extraction system for vesicles from human umbilical cord mesenchymal stem cells. The closed-loop pipeline design and continuous low-temperature control not only significantly reduce the risk of contamination but, more importantly, effectively protect the structural integrity and bioactivity of the vesicles. Redundant designs such as a precisely positionable centrifugal rotor, a liquid level detection aspiration mechanism, and mechanical limiting mechanisms ensure the reliability of the system and the consistency of product purity.

[0022] This invention provides a highly efficient and continuous extraction system for intracellular vesicles from human umbilical cord mesenchymal stem cells. This system is particularly suitable for the large-scale and standardized preparation of intracellular vesicles with stringent requirements for operating conditions and product quality, providing key technical support for the clinical translational application of vesicles in regenerative medicine, disease diagnosis, and drug delivery. Attached Figure Description

[0023] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0024] The structures, proportions, sizes, etc. illustrated in this specification are only for the purpose of assisting those skilled in the art in understanding and reading the content disclosed herein, and are not intended to limit the conditions under which the present invention can be implemented. Therefore, they have no substantial technical significance. Any modifications to the structure, changes in the proportions, or adjustments to the size, without affecting the effects and objectives that the present invention can produce, should still fall within the scope of the technical content disclosed in the present invention.

[0025] Figure 1 This is a system diagram of a highly efficient continuous extraction system for vesicles from human umbilical cord mesenchymal stem cells provided in an embodiment of the present invention. The dashed lines in the diagram represent wires, and the implementation is a pipeline. Figure 2 for Figure 1 Diagram of the mid-sonic unit structure; Figure 3 for Figure 1 Structural diagram of the centrifugal separation unit; Figure 4 for Figure 1 Structure diagram of purification unit, collection unit and control unit of medium size size exclusion chromatography column.

[0026] In the picture: 11. Ultrasonic unit; 12. Sample processing chamber; 13. Processing chamber outlet; 14. Ultrasonic generator; 15. Jacket structure; 16. Circulating cooling system; 17. Temperature sensor; 18. Processing chamber inlet; 19. One-way valve; 20. Filter; 21. Centrifugal separation unit; 22. Centrifuge chamber; 23. Centrifuge rotor; 24. Liquid inlet; 25. Supernatant outlet; 26. Replacement door; 27. Centrifuge bottle; 28. Liquid inlet needle; 29. ​​Liquid suction needle; 281. First lifting device; 291. Second lifting device; 31. Size exclusion chromatography column purification unit; 32. Size exclusion chromatography column; 33. Infusion pump; 34. Injection valve; 35. Sieve plate; 36. Mobile phase distributor; 41. Collection Unit; 51. Closed-loop piping system; 61. Control unit; 62. Central control panel; 63. Data processing module; 64. Cryogenic collection tank. Detailed Implementation

[0027] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present application, and not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present application.

[0028] like Figure 1 As shown, a highly efficient continuous extraction system for vesicles from human umbilical cord mesenchymal stem cells includes an ultrasonic unit 11, a centrifugation unit 21, a size exclusion chromatography column purification unit 31, a collection unit 41, a closed tubing system 51, and a control unit 61. The technical details of each unit are described below: 1. Ultrasonic Unit 11 The ultrasonic unit 11 described in this technology is the starting module of the entire extraction system. Its core function is to break the cell membrane through the cavitation effect and mechanical vibration of ultrasound, so that the vesicles inside the cell are released into the buffer solution outside the cell.

[0029] like Figure 2 As shown, the ultrasonic unit 11 is equipped with a sample processing chamber 12 with a water-proof function. The sample processing chamber 12 serves as a sample container and is made of a biocompatible material with a smooth inner wall to reduce non-specific adsorption of vesicles.

[0030] The sample processing chamber 12 has a processing chamber inlet 18 at its top, which is connected to a sterile tubing. A one-way valve 19 and a filter 20 are located at the processing chamber inlet 18. Samples are injected into the sample processing chamber 12 through the sterile tubing. Specifically, the processing chamber inlet 18 is the only channel for samples to enter the ultrasonic unit 11, and typically uses a Luer connector or other standard biological connector for easy connection to a syringe or other sample injection device. The one-way valve 19 is a valve that only allows fluid to flow in one direction. When a sample is injected into the sample processing chamber 12, the one-way valve 19 automatically opens; when injection stops or the pressure inside the sample processing chamber 12 is higher than the external pressure, the one-way valve 19 automatically closes to prevent sample backflow or the entry of outside air. The filter 20 is installed upstream of the one-way valve 19 and typically uses a 40μm pore size sterile filter membrane. Its function is to filter larger particulate contaminants (such as dust, fibers, etc.) from the fluid entering the sample processing chamber 12, acting as a pre-filter to protect downstream, more delicate components. In pneumatically driven sample transfer methods, filter 20 also filters the driving gas to prevent particulate contaminants from entering the sample flow path with the gas. This technology constructs a one-way pure sample input channel by setting a one-way valve 19 and filter 20 at the processing chamber inlet 18 of the sample processing chamber 12. This prevents backflow loss of the sample during processing and also provides necessary purification assurance for pneumatically driven sample transfer methods. This is of great significance for maintaining the overall system status and sample integrity.

[0031] The sample processing chamber 12 has a processing chamber outlet 13 at its bottom to facilitate the complete discharge of the broken cell suspension. The processing chamber outlet 13 is connected to a medical-grade silicone tube with a smooth inner wall. This silicone tube passes through an electric clamp valve or a solenoid valve and is then connected to the feed inlet of the centrifuge unit 21. A separate waste liquid discharge line is also provided at the bottom of the sample processing chamber 12 for discharging waste liquid during system cleaning.

[0032] The ultrasonic generator 14 is the core component for generating ultrasonic waves. Its operating frequency is typically in the range of 20 kHz to 100 kHz, preferably 40 kHz, and its power range is 10-100 W, adjustable according to the volume of the sample and the required fragmentation. Specific pulse mode parameters are: 2 seconds of operation followed by a 3-second interval, with the total processing time adjusted to 3-10 minutes depending on the sample volume. The ultrasonic generator 14 is connected via cable to an ultrasonic transducer (oscillator) installed at the bottom or side wall of the sample processing chamber 12. Acoustic coupling is achieved through thermally conductive silicone grease or direct contact, converting electrical energy into high-frequency mechanical vibration.

[0033] The sample processing chamber 12 employs a jacket structure 15 for circulating coolant. The inner layer is a sample container (volume 1-10 mL, customizable), and the outer layer is a circulating coolant jacket. The jacket structure 15 connects to a circulating cooling system 16, which typically includes a refrigeration compressor, a heat exchanger, a coolant storage tank, and a circulating pump. It provides a coolant with a stable temperature, typically deionized water or an ethylene glycol aqueous solution at 4°C, with a temperature control range of 2-8°C. The circulating pump is preferably a miniature magnetic pump with a flow rate range of 10-100 mL / min, adjustable according to cooling requirements.

[0034] The jacket device is equipped with a temperature sensor 17 to ensure that the ultrasonic process is carried out at a low temperature. The temperature sensor 17 is a high-precision platinum resistance thermometer (Pt100) or thermocouple with a measurement accuracy of ±0.1℃. It monitors the temperature of the coolant in the jacket in real time and feeds the signal back to the control unit 61. When the temperature exceeds the set threshold, the control unit 61 automatically adjusts the cooling power of the circulating cooling system 16 or increases the coolant flow rate. A PID control algorithm is used for precise temperature control to ensure that the ultrasonic crushing process is always carried out under low-temperature conditions.

[0035] By integrating a jacket structure 15 and a temperature sensor 17 monitoring system into the ultrasonic unit 11, the problem of sample temperature rise caused by the conversion of ultrasonic energy into heat during traditional ultrasonic disruption is effectively solved. The low-temperature environment not only protects the integrity of the vesicle membrane, preventing its aggregation or rupture due to thermal stress, but also inhibits protein degradation and enzyme activity, maintaining the bioactivity of the vesicle contents. This is crucial for obtaining high-quality, highly active intracellular vesicle products.

[0036] 2. Centrifugal Separation Unit 21 The centrifugal separation unit 21 described in this technology is the core separation module of the system. It uses centrifugal force to precipitate large particles such as cell debris and organelles to the bottom of the centrifuge bottle 27, while the supernatant containing vesicles is retained in the upper layer. The centrifugal separation unit 21 includes a centrifuge chamber 22, a rotatable centrifuge rotor 23, a liquid inlet module, a supernatant outlet module, and a replacement door 26 located on the side wall of the centrifuge chamber 22.

[0037] like Figure 2As shown, the centrifuge rotor 23 is equipped with at least one centrifuge bottle 27 with its opening facing upwards. The centrifuge bottle 27 is a disposable sterile plastic bottle, commonly available in 50 mL or 100 mL sizes, and the bottle body has sufficient strength to withstand the centrifugal force generated by high-speed centrifugation. The centrifuge bottle 27 is sealed with a self-sealing rubber diaphragm made of medical-grade silicone rubber or butyl rubber, possessing good elasticity and sealing performance. When the inlet needle 28 or aspiration needle 29 punctures the diaphragm, the diaphragm tightly wraps around the needle, forming a dynamic seal to prevent liquid leakage or the entry of outside air. When the needle is withdrawn, the elasticity of the diaphragm allows it to automatically close, restoring the seal, and maintaining good sealing performance even after multiple punctures. The rubber diaphragm is usually integrated with the plastic bottle cap through injection molding or bonding; during use, simply screw the bottle cap onto the centrifuge bottle 27. This technology achieves complete sample sealing during centrifugation by using a centrifuge bottle 27 with a self-sealing rubber diaphragm. Throughout the entire process—from sample entry into centrifuge vial 27, centrifugation, and supernatant aspiration—centrifuge vial 27 remains sealed, eliminating the need for opening. This is crucial for maintaining sample sterility and preventing the loss of volatile components. Furthermore, the disposable nature of the vial 27 avoids the risk of cross-contamination and simplifies equipment cleaning and validation.

[0038] The centrifuge chamber 22 is a closed cavity, capable of maintaining a low-temperature environment and preventing sample contamination. A manually operable replacement door 26 is located on the side wall or top of the centrifuge chamber 22. The replacement door 26 is equipped with a safety interlock switch; the centrifuge cannot start when the door is open. Specifically, when the door is open, the centrifuge power is cut off, and the inlet needle 28 / absorption needle 29 is prevented from descending; if centrifugation has not stopped, the door lock electromagnet is engaged, preventing it from opening. The safety interlock switch is either photoelectric or mechanical, and the signal is transmitted to the control unit 61 via a digital I / O interface.

[0039] The liquid inlet module is connected to the liquid outlet 13 of the processing chamber. The liquid inlet module is equipped with a movable liquid inlet needle 28, which is mounted on a first lifting device 281. The movable liquid inlet needle 28 of the liquid inlet module is located inside the centrifuge chamber 22. The first lifting device 281 can be any of the following structures: electric push rod, lead screw assembly, or gear rack assembly. In this technology, the first lifting device 281 is preferably an electric push rod, which has a simple structure and low cost. The liquid inlet needle 28 is made of stainless steel or other biocompatible materials, and its tip is sharp and smooth, making it easy to pierce the sealing diaphragm of the centrifuge bottle 27.

[0040] The supernatant discharge module is equipped with a supernatant discharge port 25 and a vertically movable suction needle 29, which is located inside the centrifuge chamber 22. The supernatant discharge module is mounted on a second lifting device 291, which can be any of the following structures: an electric push rod, a lead screw assembly, or a gear and rack assembly. Preferably, a high-precision stepper motor combined with a lead screw assembly is used to facilitate the slow descent of the suction needle 29 during suction, preventing it from inserting into the sediment. The suction needle 29 is made of stainless steel or other biocompatible materials, with a sharp and smooth tip for easy piercing of the sealing diaphragm of the centrifuge bottle 27.

[0041] The centrifugal rotor 23 is driven by a servo motor, which is the power source for rotating the centrifugal rotor 23. Compared with ordinary asynchronous motors, servo motors have advantages such as high speed control accuracy, fast response speed, and good torque characteristics. The servo motor is preferably 200 W in power, with a maximum speed of 10,000 rpm, and can provide a maximum centrifugal force of 10,000 × g. The low-temperature control function is achieved by setting a cooling jacket or refrigeration coil around the motor and the centrifuge chamber 22, which can stably control the temperature inside the centrifuge chamber 22 at about 4°C. This is crucial for protecting the biological activity of vesicles, because low temperature can reduce enzyme activity and molecular motion rate, reducing vesicle aggregation and degradation.

[0042] The rotor is precisely positioned using a high-resolution encoder with a resolution of up to 10,000 pulses per revolution and a positioning accuracy of ±0.1°. A homing operation is performed after each power-on, using a photoelectric sensor or Hall effect sensor as the origin marker to eliminate accumulated errors. Position control employs a PID algorithm to ensure the rotor stops accurately at a preset angle. Through these position detection elements, the control unit 61 can precisely control the rotor's stopping angle, ensuring the centrifuge bottle 27 is accurately aligned with the inlet needle 28, suction needle 29, or replacement door 26.

[0043] This technology preferably utilizes a servo motor with position feedback to drive the centrifuge rotor 23, achieving seamless switching between two operating modes: high-speed centrifugation and precise positioning. During centrifugation, the high-speed rotation of the motor generates sufficient centrifugal force to achieve solid-liquid separation; after centrifugation, the motor can quickly decelerate and precisely stop at a preset angle, aligning the centrifuge bottle 27 with the next operating station. This integrated design eliminates the manual positioning step required in traditional centrifuges, greatly improving automation and operational efficiency. Simultaneously, continuous low-temperature control provides ongoing protection for the bioactivity of the samples.

[0044] To ensure that the aspiration needle 29 does not enter the sediment layer, the following structural improvements are made to this technology: The lifting mechanism of the aspiration needle 29 employs a high-precision stepper motor combined with a lead screw assembly, achieving a resolution of 0.01 mm / step. The lifting mechanism of the aspiration needle 29 is equipped with upper and lower limit positions. The upper limit position is the highest position the aspiration needle 29 can reach, typically positioned sufficiently above the mouth of the centrifuge bottle 27 to ensure no collision occurs during rotor rotation. The lower limit position is positioned 1-2 mm from the bottom of the centrifuge bottle 27, serving as a final protection against electrical failure and ensuring the aspiration needle 29 does not contact the bottom of the bottle. Both the upper and lower limit positions of the lifting mechanism can utilize a combination of an adjustable screw, a limit stop, and a locking nut, allowing for adjustable height of the limit stop with an adjustment accuracy of 0.1 mm.

[0045] The lower limit position is the lowest position that the aspiration needle 29 can descend to, and this position needs to be precisely adjusted according to the specifications of the centrifuge bottle 27. In actual operation, the lower limit position is first adjusted to 1-2 mm from the bottom of the bottle according to the specifications of the centrifuge bottle 27, serving as a mechanical hard limit. This mechanical limit is a hard limit, which can forcibly prevent the aspiration needle 29 from continuing to descend even in extreme cases such as electrical control system failure, sensor failure, or program error, physically ensuring that the aspiration needle 29 will not insert into the sediment layer. The mechanical limit is set 0.2-0.5 mm below the electrical limit and is triggered only in the event of electrical failure. A soft limit is set in the software, 0.3 mm higher than the mechanical limit, forming triple protection. The actual liquid level control is achieved by real-time detection by a capacitive liquid level sensor.

[0046] The lower limit position is adjusted to a set distance above the theoretical top surface of the sediment layer at the bottom of the centrifuge bottle 27. This distance is determined based on empirical values ​​or pre-experiments, such as approximately 2-3 mm. This structure, through mechanical means, ensures that the lowest descent position of the aspiration needle 29 is always above the sediment layer. Even if all sensors fail, the mechanical limit can forcibly stop the aspiration needle 29, preventing it from inserting into the sediment layer. Based on the dimensions of the centrifuge bottle 27 and the height tolerance of the rotor station, the number of pulses required to descend from the upper limit position to the "safe contact liquid surface" and "absorption endpoint" is pre-calibrated. During actual operation, the controller sends a precise number of pulses to drive the aspiration needle 29 to the target height.

[0047] The lower limit position is crucial. This technology can be achieved by improving the centrifuge bottle 27 by adding a concave structure at the center of its bottom. This concave structure is an upwardly convex circular platform with a diameter matching the outer diameter of the aspiration needle 29. The height is empirically set at 1.5-2 mm, slightly higher than the expected height of the precipitate. When the aspiration needle 29 descends to a certain point, it will encounter the concave structure, at which point it can no longer descend, thus achieving the lower limit position in terms of physical structure. The tip of the aspiration needle 29 is designed to be flat or round to avoid damage to the needle tip or the generation of particles upon impact. A pressure sensor or overcurrent detection is added to the lifting mechanism of the aspiration needle 29; it stops upon detecting a contact.

[0048] To more accurately detect the liquid level and eliminate errors, this technology employs a liquid level sensor as the core solution to the problem of "inconsistent liquid level height." This sensor is adaptive to different liquid levels, unaffected by liquid color or transparency, and exhibits high sensitivity. This technology can utilize any of the following: capacitive liquid level sensor, photoelectric liquid level sensor, pressure liquid level sensor, or ultrasonic liquid level sensor. Preferably, a capacitive liquid level sensor is used. Specifically, an insulating layer is wrapped around the aspiration needle 29 (made of stainless steel) to form a capacitor. The needle body of the aspiration needle 29 serves as one electrode, and the liquid it contacts (conductive) serves as the other electrode.

[0049] The insulating layer is coated with parylene C, with a thickness of 5-10 μm, ensuring insulation performance without affecting capacitance detection sensitivity. The insulating material must comply with ISO 10993 biocompatibility standards. The capacitance detection circuit uses a dedicated capacitance detection chip such as the FDC2214 or AD7746, a high-resolution capacitance-to-digital converter (CDC), with a resolution reaching the fF level. Detection sensitivity can reach 0.1 pF, with a response time of less than 10 ms. The capacitance change threshold is experimentally set to be greater than 5 pF as the liquid surface contact threshold. Digital filtering algorithms (such as moving average and Kalman filtering) are used to eliminate noise interference, and automatic baseline calibration is performed before each use. An alternative solution is to add a photoelectric liquid level detector as redundancy.

[0050] The aspiration needle 29 descends at a constant speed of 0.5 mm / s. When the tip of the needle 29 contacts the liquid surface, the capacitance value changes abruptly. The controller immediately records the current position pulse count and stops descending. Then, a descent depth is set from the stop position (e.g., the needle tip descends another 1.5 mm), and aspiration begins. During aspiration, the liquid level will drop, but since the flow rate is known, the capacitance change can be monitored by timing or in real time. If the needle tip leaves the liquid surface, the capacitance value rebounds (the threshold is set to be greater than 3 pF). The controller then sends pulses to the stepper motor to descend the aspiration needle 29 in 0.1 mm steps, ensuring that the needle tip is always submerged in the supernatant. The descent compensation time interval is 100 ms.

[0051] Even if the liquid level sensor locates the initial liquid level, it is still necessary to prevent the needle tip from entering the sediment layer during liquid aspiration. This technique sets the initial liquid level height as H0, the descent depth of the aspiration needle 29 after contacting the liquid surface as d (e.g., 2 mm), and then begins liquid aspiration. The preset aspiration flow rate v (mL / s) of the aspiration needle 29 and the total volume of the supernatant V allow for the estimation of the aspiration time t ≈ V / v. A stepped deceleration aspiration method is used: the flow rate is reduced by 50% near the bottom. When aspiration reaches t - Δt, the aspiration needle 29 is actively raised a certain distance (e.g., 0.5 mm) to avoid bottom eddy disturbance of the sediment; the remaining 50-100 μL of supernatant is not aspirated to ensure no disturbance of the sediment. A side-hole design for the aspiration needle 29 can be added: openings on the side of the needle tip to avoid vertical upward suction force; and a buffer structure is added to the end of the aspiration needle 29 to reduce fluid shear force.

[0052] While the centrifugation unit of this technology ensures that large precipitates are not aspirated, it is impossible to completely avoid the aspiration of trace amounts of fine particles. Fragments larger than 200 nm may remain suspended and elute simultaneously with the vesicles in the pore volume. However, for vesicle extraction, the subsequent size exclusion chromatography column purification unit 31 will further separate small molecule impurities such as proteins and nucleic acids; therefore, a small amount of aspiration will not affect the purity of the final product. A filtration step (e.g., a 0.22 μm filter) can be added between the centrifugation and chromatography units to further improve purity. Post-chromatographic purity verification methods (e.g., nanoparticle tracking analysis (NTA)) can also be added.

[0053] 3. Size exclusion chromatography column purification unit 31 The size exclusion chromatography column purification unit 31 utilizes the principle of gel filtration to separate vesicles from impurities such as proteins and nucleic acids based on their molecular size. The size exclusion chromatography column purification unit 31 is connected to the supernatant outlet 25 of the centrifugation unit 21 for separation and enrichment based on vesicle particle size. The supernatant outlet 25 is connected to the quantitative loop of the injection valve 34 via tubing, and the infusion pump 33 provides the power for sample loading.

[0054] like Figure 4 As shown, the size exclusion chromatography column 32 of the size exclusion chromatography column purification unit 31 uses glass or biocompatible polymer column tubes. Glass column tubes have advantages such as high transparency, good chemical stability, and smooth inner walls, facilitating observation of the column bed state and sample separation process, and are commonly used column tube materials in laboratories. Biocompatible polymer column tubes (such as polypropylene, polyetheretherketone, etc.) have advantages such as light weight, durability, and resistance to acid and alkali corrosion, making them more suitable for industrial-scale applications.

[0055] The column is preferably 19 mm in inner diameter and 100 mm in length, with a sample loading volume of 0.5 mL; alternatively, it can have an inner diameter of approximately 38 mm, a height of approximately 180 mm, and a sample loading volume of 10 mL. The column packing material is an agarose or dextran-based size exclusion medium with a size exclusion limit of 35 nm and a separation range of 30-200 nm. The column dimensions are optimized based on the volume of the sample to be separated and the separation requirements. Columns with an inner diameter of 15 mm to 40 mm are preparative or semi-preparative columns, capable of handling milliliter-level sample volumes, matching the processing capacity of the centrifugation unit. A column length of 100 mm to 200 mm provides sufficient separation path length to ensure adequate separation of molecules of different sizes. The agarose-based size exclusion medium (such as the Sepharose series) has good chemical stability and mechanical strength, uniform pore size distribution, and low non-specific adsorption, making it ideal for the separation of biomolecules and nanoscale vesicles. Size exclusion media with a dextran matrix (such as the Sephadex series) have higher specific surface area and separation capacity, making them suitable for separating substances with small molecular weight differences. Based on the particle size distribution of intracellular vesicles (typically between 30 nm and 200 nm), media with a size exclusion limit of approximately 35 nm and a separation range covering 30 nm to 200 nm are most suitable. This ensures that vesicles are excluded from the packing particles and eluted in the interstitial volume, while smaller impurities such as proteins and nucleic acids (less than 35 nm) enter the packing particles and are eluted later, thus achieving effective separation of vesicles and impurities. Fragments larger than 200 nm will elute along with vesicles in the interstitial volume, potentially affecting purity and requiring thorough removal during the preceding centrifugation step.

[0056] The column is equipped with replaceable sieve plates 35 and mobile phase distributors 36 at both ends. The sieve plates 35, typically made of porous sintered material (such as polyethylene or stainless steel sintered mesh), are installed at both ends of the column. The sieve plates 35 preferably have a pore size of 10 μm, smaller than the particle size of the chromatographic packing material. Their function is to prevent packing particles from flowing out of the column while allowing liquid and the substances to be separated to pass freely. The mobile phase distributor 36 is installed at the inlet end of the column and employs an umbrella-shaped flow channel design. Its function is to evenly distribute the sample solution entering the column across the entire cross-section of the column bed, preventing the sample from concentrating at the center of the column, which would reduce column efficiency and resolution.

[0057] The chromatographic operating parameters are as follows: the mobile phase uses PBS buffer (pH 7.4), the flow rate ranges from 0.5 to 2 mL / min, and the injection volume is recommended to be 1-5% of the column volume. The column pressure is typically in the range of 0.5-5 bar. The relationship between elution volume and peak time is as follows: the elution time corresponding to the void volume (approximately 30% of the column volume) is the vesicle elution peak, such as 12-18 minutes (at a flow rate of 1 mL / min). The infusion pump 33 uses a constant flow plunger pump with a flow rate accuracy of ±1%. The quantitative loop volume of the injection valve 34 is selected according to the required injection volume, such as 0.5, 1, or 2 mL, and the switching control method is solenoid valve driven, automatically controlled by the control unit 61.

[0058] This technology significantly improves the efficiency and reproducibility of chromatographic separation by optimizing the structural design of the size exclusion column 32, particularly by employing replaceable sieve plates 35 and a high-efficiency mobile phase distributor 36. The uniform sample distribution fully utilizes the separation capacity of the entire column bed, increasing the theoretical plate number of the column and thus achieving better resolution, enabling more effective separation of vesicles and impurities. The replaceable sieve plate 35 design also facilitates column maintenance and regeneration, extending the column's lifespan.

[0059] 4. Collection Unit 41 The collection unit 41 is used to collect the target vesicle components after chromatographic purification and to preserve them at low temperature to maintain their biological activity. The collection unit 41 is connected to the size exclusion chromatography column purification unit 31 to collect the final product.

[0060] The collection unit 41 employs a multi-channel automated collector, which features a rotating collection tray holding multiple collection tubes (e.g., 5 mL EP tubes) for collecting the target elution peak. The outlet of the size exclusion column 32 is connected to the inlet three-way valve of the multi-channel automated collector (which can be switched to waste liquid or collection tubes). Each collection tube of the automated collector has a movable dispensing needle above it, through which the target eluent flows into the selected collection tube. The collection tubes are then connected to a low-temperature collection tank 64 via a main pipe. The low-temperature collection tank 64 is equipped with a double jacket and can be pre-cooled to 4°C. The tank body has a sealing cap and a discharge port. All piping is a closed system, with the valve switching and collection tray rotation coordinated by the control unit 61.

[0061] 5. Closed-loop piping system 51 The closed pipeline system 51 includes connecting pipelines between the above-mentioned units and a liquid delivery drive device, which is used for the continuous flow of the sample in a closed environment, organically connecting the various units to form a complete closed flow path. The sample is always in a closed environment throughout the entire processing, which greatly reduces the risk of contamination.

[0062] The closed-loop piping system 51 includes medical-grade silicone tubing, such as medical-grade platinum-cured silicone tubing or PTFE tubing, connecting the processing chamber outlet 13 to the inlet device of the centrifugal separation unit 21, the supernatant outlet 25 to the size exclusion chromatography column 32, and the size exclusion chromatography column 32 to the collection unit 41. The medical-grade silicone tubing has a smooth inner wall and low adsorption. The medical-grade silicone tubing is manufactured using a platinum curing process, exhibiting excellent biocompatibility, temperature resistance, and chemical stability. Its inner wall undergoes special treatment to achieve extremely high smoothness, significantly reducing non-specific adsorption of biomolecules and vesicles on the tubing wall and improving sample recovery. The inner diameter of the silicone tubing is typically between 0.5 mm and 5 mm, and can be selected based on the fluid flow rate and sample volume.

[0063] Sample transfer is primarily pneumatically driven, supplemented by a miniature peristaltic pump (for transporting the supernatant to the column). The pneumatic drive is implemented as follows: the pressure source is a miniature air pump or external compressed air, with a pressure range of 0.05-0.2 MPa. A precision pressure regulating valve is used for pressure adjustment, with a pressure stabilization accuracy of ±0.01 MPa. The driving gas must be filtered through a 0.22 μm filter. The sequence of pneumatic drive and the electric clamp valve is as follows: open the valve first, then pressurize; after transfer, depressurize first, then close the valve. Transfer time: For example, the transfer time for 1 mL of sample at 0.2 MPa pressure through a 1 mm inner diameter, 500 mm long tube is approximately 5-10 seconds.

[0064] The medical-grade silicone tubing passes through an electric clamp valve or a solenoid valve and connects to the liquid inlet module of the centrifugation unit 21. Key components include an electric clamp valve, a check valve, and a pressure sensor. The electric clamp valve controls fluid flow by squeezing or releasing the tubing. Its advantage is that it does not directly contact the sample, avoiding cross-contamination, and is particularly suitable for processing biological samples with high sterility requirements. The solenoid valve controls the fluid passage by driving the valve core with electromagnetic force, offering fast response and high control precision. Both valves are controlled by electrical signals from the control unit 61, enabling precise timing of switching. The micro-peristaltic pump has a flow rate range of 0.1-5 mL / min, and its operation with the supernatant delivery is as follows: the peristaltic pump starts when aspiration begins and stops when aspiration ends.

[0065] The closed-loop piping system 51 uses medical-grade silicone tubing as the fluid transport channel, combined with an electric clamp valve or solenoid valve for precise fluid control, to construct a completely closed sample processing flow path. From the moment the sample enters the ultrasonic unit 11 until it is finally collected in the cryogenic collection container 64, it remains in a closed piping environment, without contact with outside air, and requires no manual opening, fundamentally eliminating the possibility of external contamination and avoiding sample loss during transfer.

[0066] 6. Control Unit 61 The control unit 61 is connected to all the aforementioned units and is used to control the entire process operation. The control unit 61 includes a central control panel 62 and a data processing module 63. The central control panel 62 is equipped with a touchscreen, which allows for programmable settings of ultrasonic parameters, centrifugation parameters, and chromatographic operating parameters. Specific settable parameter ranges include: ultrasonic power (10-100 W), ultrasonic time (1-30 min), centrifugation speed (1000-10000 rpm), centrifugation time (1-60 min), and chromatographic flow rate (0.1-5 mL / min). The displayed information includes real-time temperature, pressure, speed, liquid level, and operating status.

[0067] The data processing module 63 is used to record signals from various sensors (temperature, pressure, liquid level, rotation speed, rotor position, etc.) to achieve process monitoring and alarms. The sensor signal acquisition frequency is 10 Hz. Specific parameters recorded include: complete operating parameters for each sample (ultrasonic power / time, centrifugation speed / time, chromatographic flow rate / pressure, etc.), alarm threshold settings (temperature over-limit, pressure over-limit, liquid level detection failure, etc.), and data storage method: local SD card or cloud storage.

[0068] 7. The workflow of this technology is as follows (taking a single sample as an example, multiple samples can be processed in cycles): During the preparation phase, the replacement door is manually opened, a sterile centrifuge bottle is placed in, and the door is closed. Once the system detects that the door is closed and the interlock is active, it enters standby mode.

[0069] During the cell suspension input stage, approximately A single-cell suspension (approximately 10 mL) made from umbilical cord mesenchymal stem cells is injected into the sample processing chamber of the ultrasound unit through the processing chamber inlet. A one-way valve and filter ensure that the injection process is sterile.

[0070] During the ultrasonic disruption phase, ultrasonic parameters are set on the touchscreen of the control unit, such as power 50 W, pulse mode (2 seconds on, 3 seconds off), total time 5 minutes, and a circulating cooling system maintaining a low temperature of 4°C. After ultrasonication, the cells are disrupted, and intracellular vesicles are released into the supernatant.

[0071] During the automatic sample introduction stage, the control unit issues a command, and the servo motor drives the centrifuge rotor to rotate to the liquid inlet position. The liquid inlet needle descends under the drive of the electric push rod, piercing the self-sealing rubber diaphragm of the centrifuge bottle. Then, the electric clamp valve at the bottom of the ultrasonic unit opens, and the suspension flows into the centrifuge bottle through the medical-grade silicone tube under air pressure.

[0072] After the liquid inlet is complete, the inlet needle rises to its original position and the valve closes. During the centrifugation stage, the rotor is rotated to any position, and the centrifugation program is started with parameters set to 5000×g, centrifugation time 15 minutes, and temperature 4℃. During this process, large particles such as cell debris and organelles settle to the bottom of the centrifuge bottle under centrifugal force.

[0073] During the supernatant aspiration stage, after centrifugation stops, the rotor is precisely positioned at the aspiration station, and the aspiration needle descends slowly at a speed of 0.5 mm / s, driven by a high-precision lead screw assembly. When the needle tip contacts the liquid surface, the capacitive level sensor detects a sudden change in capacitance (threshold > 5 pF), and the control unit records the current position and temporarily stops the descent. The needle then continues to descend to a preset depth (e.g., 1.5 mm) before aspiration begins. During aspiration, the control unit estimates the aspiration time based on the preset flow rate and total volume. Near the end of aspiration, it actively raises the needle a short distance, employing a stepped deceleration aspiration method to avoid bottom eddy disturbance and sedimentation. The aspirated supernatant (approximately 0.5-0.8 mL, with a final residual 50-100 μL not aspirated) is delivered to the quantitative loop of the chromatographic unit's injection valve through the supernatant discharge valve. After aspiration is complete, the needle rises back to its original position.

[0074] During the chromatographic purification stage, the infusion pump injects PBS buffer (pH 7.4) at a constant flow rate (e.g., 1 mL / min) to wash the column, and then the sample in the quantitative loop of the injection valve is pushed into the size exclusion column. According to the pre-calibrated peak time (e.g., 12-18 minutes for vesicle elution), when the target component is about to elute, the multi-channel autocollector switches to collection mode.

[0075] During the collection and preservation stage, the target eluent flows into a cryogenic collection tank via an automated collector and is stored at 4°C to maintain the biological activity of the vesicles.

[0076] During the centrifuge bottle replacement stage, the rotor rotates to the replacement station, the replacement door is manually opened, the used centrifuge bottle (containing sediment) is taken out, a new sterile centrifuge bottle is placed in, and after the door is closed, the system automatically detects and confirms that the safety interlock is effective.

[0077] To process the next sample, repeat steps 2 through 9 above if more samples need to be processed, enabling continuous operation. The total time for a single sample is approximately 60 minutes, with manual bottle changing taking only about 30 seconds, which does not affect the continuous processing of multiple samples.

[0078] Those skilled in the art will clearly understand that, for the sake of convenience and brevity, the specific working process of the systems, devices, apparatuses, modules or units described above can be referred to the corresponding process in the foregoing method embodiments, and will not be repeated here.

[0079] It should be understood that in this application, "at least one (item)" means one or more, and "more than" means two or more. "And / or" is used to describe the relationship between related objects, indicating that three relationships can exist. For example, "A and / or B" can represent three cases: only A exists, only B exists, and both A and B exist simultaneously, where A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects are in an "or" relationship. "At least one (item) of the following" or similar expressions refer to any combination of these items, including any combination of single or plural items. For example, at least one (item) of a, b, or c can represent: a, b, c, "a and b", "a and c", "b and c", or "a and b and c", where a, b, and c can be single or multiple.

[0080] In the several embodiments provided in this application, it should be understood that the disclosed systems, apparatuses, and methods can be implemented in other ways. For example, the apparatus embodiments described above are merely illustrative; for instance, the division of units is only a logical functional division, and in actual implementation, there may be other division methods. For example, multiple units or components may be combined or integrated into another system, or some features may be ignored or not executed. Furthermore, the coupling or direct coupling or communication connection shown or discussed may be an indirect coupling or communication connection between apparatuses or units through some interfaces, and may be electrical, mechanical, or other forms.

[0081] The units described as separate components may or may not be physically separate. The components shown as units may or may not be physical units; that is, they may be located in one place or distributed across multiple network units. Some or all of the units can be selected to achieve the purpose of this embodiment according to actual needs.

[0082] Furthermore, the functional units in the various embodiments of this application can be integrated into one processing unit, or each unit can exist physically separately, or two or more units can be integrated into one unit. The integrated unit can be implemented in hardware or as a software functional unit.

[0083] If the integrated unit is implemented as a software functional unit and sold or used as an independent product, it can be stored in a computer-readable storage medium. Based on this understanding, the technical solution of this application, in essence, or the part that contributes to the prior art, or all or part of the technical solution, can be embodied in the form of a software product. This computer software product is stored in a storage medium and includes several instructions to cause a computer device (which may be a personal computer, server, or network device, etc.) to execute all or part of the steps of the methods described in the various embodiments of this application. The aforementioned storage medium includes: USB flash drives, portable hard drives, read-only memory (ROM), random access memory (RAM), magnetic disks, optical disks, and other media capable of storing program code.

[0084] The above-described embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. A highly efficient continuous extraction system for vesicles from human umbilical cord mesenchymal stem cells, characterized in that, include: An ultrasound unit is used to break up cells to release intracellular vesicles. The ultrasound unit is equipped with a sample processing chamber with a water-proof function, and the bottom of the sample processing chamber is equipped with a liquid outlet. The centrifugal separation unit includes a centrifuge chamber, a rotatable centrifugal rotor, a liquid inlet module, a supernatant outlet module, and a replacement door located on the side wall of the centrifuge chamber. The centrifugal rotor is equipped with at least one centrifuge bottle with its opening facing upwards. The liquid inlet module is connected to the liquid outlet of the processing chamber and is equipped with a liquid inlet needle that can be raised and lowered. The supernatant outlet module is equipped with a supernatant outlet and a liquid suction needle that can be raised and lowered. The liquid inlet needle, liquid suction needle, and centrifugal rotor are all located inside the centrifuge chamber. The centrifugal rotor can rotate and position the centrifuge bottle directly below the liquid inlet needle or the liquid suction needle, or at the replacement door. The size exclusion chromatography column purification unit is connected to the supernatant outlet of the centrifugation separation unit and is used for separation and enrichment based on vesicle particle size. The collection unit is connected to the size exclusion chromatography column purification unit to collect the final product; A closed-loop piping system, including the connections between the above-mentioned units and the provision of liquid transport power, is used for the continuous flow of samples in a closed environment; The control unit, connected to all the above units, is used to control the entire process operation.

2. The efficient continuous extraction system for vesicles from human umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The ultrasonic unit is equipped with an ultrasonic generator that applies ultrasonic waves to the sample processing chamber. The outer layer of the sample processing chamber is equipped with a jacket structure for circulating coolant. The jacket structure is connected to a circulating cooling system. A temperature sensor is installed inside the jacket to ensure that the ultrasonic process is carried out at a low temperature.

3. The efficient continuous extraction system for vesicles from human umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The closed piping system includes a smooth-walled medical-grade silicone tube connected to the liquid outlet of the treatment chamber. The medical-grade silicone tube passes through an electric clamp valve or a solenoid valve and is then connected to the liquid inlet module of the centrifugal separation unit.

4. The efficient continuous extraction system for vesicles from human umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The inlet needle and the suction needle are moved up and down by any one of the following structures: electric push rod, lead screw assembly, and gear rack assembly.

5. The efficient continuous extraction system for vesicles from human umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The centrifuge bottle is sealed with a self-sealing rubber diaphragm.

6. The efficient continuous extraction system for vesicles from human umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The lifting mechanism of the aspiration needle has an upper limit position and a lower limit position. The lower limit position is adjusted to a set distance above the top surface of the theoretical sediment layer at the bottom of the centrifuge bottle to ensure that the aspiration needle will not enter the sediment layer.

7. The efficient continuous extraction system for vesicles from human umbilical cord mesenchymal stem cells according to claim 6, characterized in that: The centrifuge bottle has a concave structure at the bottom center, and the height of the concave structure is slightly higher than the height of the precipitate. When the aspiration needle descends to a certain extent, it will encounter the concave structure, at which point the aspiration needle can no longer move down.

8. The efficient continuous extraction system for vesicles from human umbilical cord mesenchymal stem cells according to claim 6, characterized in that: The aspiration needle is covered with an insulating layer, forming a capacitor. The needle body acts as one pole, and the liquid it contacts acts as the other pole. When the needle tip touches the liquid surface, the capacitance value changes abruptly. The controller immediately records the current position pulse count and stops descending. Then, a descent depth is set from the stop position, and aspiration begins. During the aspiration process, the capacitance change is monitored in real time. If the capacitance value rebounds, the controller sends a pulse to the stepper motor to lower the needle, ensuring that the needle tip is always immersed in the supernatant.

9. The efficient continuous extraction system for vesicles from human umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The size exclusion chromatography column purification unit uses a glass or biocompatible polymer column tube, with replaceable sieve plates and mobile phase distributors at both ends of the column tube.

10. The efficient continuous extraction system for vesicles from human umbilical cord mesenchymal stem cells according to claim 9, characterized in that: The size exclusion chromatography column has an inner diameter ranging from 15 to 40 mm and a length ranging from 100 to 200 mm. The packing material inside the size exclusion chromatography column is a size exclusion medium based on agarose or dextran, with an exclusion limit of 35 nm and a separation range of 30 to 200 nm.

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