Adipose-derived stem cell exosome culture device and culture method

Abstract

The invention belongs to the technical field of adipose-derived stem cell culture, and discloses an adipose-derived stem cell exosome culture device and a culture method.The adipose-derived stem cell exosome culture device comprises a culture box, a water bath box and a power control box which are two independent sealing boxes are arranged in the culture box, and a first sliding frame is slidably installed at the position, close to the upper end, in the water bath box; a plurality of mounting plates which are uniformly distributed at intervals are fixedly mounted on the first sliding frame, a constant temperature device is arranged on the inner wall, located below the mounting plates, of the water bath box, a double-output-shaft motor is fixedly mounted at the position, away from the water bath box, in the power control box, and the double-output-shaft end of the double-output-shaft motor is in transmission connection with a multi-connecting-rod device; the other end of the multi-connecting-rod device is fixedly connected with the first sliding frame; the whole structure is simple, the temperature control precision can be improved in the adipose-derived stem cell exosome culture process, batch culture can be achieved, the problems that adipose tissue blocks make contact with digestive enzymes unevenly, and the cell extraction rate is low are solved, and the use effect is improved.

Description

Technical Field

[0001] This invention belongs to the field of adipose stem cell culture technology, specifically, it relates to an adipose stem cell exosome culture device and culture method. Background Technology

[0002] Stem cell exosomes, as tiny vesicles with a diameter of 40-100 nm, can carry biomolecules such as lipids, proteins, and mRNA to achieve intercellular communication, and have important application value in tissue repair, anti-inflammation, and anti-fibrosis. Adipose-derived stem cells (ADSCs) are the preferred cell source for exosome preparation due to their abundant availability, convenient acquisition, and high exosome secretion. Efficient culture devices and methods are the core guarantee for improving the extraction rate of ADSCs and the yield of exosomes.

[0003] In the culture of adipose-derived stem cells via exosomes, mechanical movement is necessary to ensure sufficient contact between the adipose tissue mass and digestive enzymes, promoting the release of stem cells from the tissue. Simultaneously, precise temperature control is crucial to maintain enzyme activity and cell stability. Traditional culture methods often rely on manual shaking of the culture dish, which is not only labor-intensive but also suffers from uneven shaking frequency and insufficient contact, resulting in low stem cell extraction efficiency and impaired cell viability. To address this issue, automated shaking culture devices have emerged in related technological fields.

[0004] Chinese patent application number CN202211233827.8 discloses an adipose-derived stem cell exosome culture device. It solves the technical problem of traditional adipose-derived stem cell exosome culture devices being unable to agitate the culture dishes during the culture process. The device includes a culture chamber with a hinged door at the front end, a power mechanism on the side, and a rotating shaft rotatably connected inside the chamber. The upper end of the shaft passes through the upper end of the chamber, and a rotating mechanism is provided on the circumference of the shaft to rotate the culture dishes. A placement slot is connected to the shaft via the rotating mechanism, and the culture dishes are placed in the slot. This invention can agitate the culture dishes in the placement slot, thus achieving the purpose of shaking the culture dishes; this invention is beneficial for the culture of adipose-derived stem cell exosomes and reduces the labor intensity of workers. However, the aforementioned existing patents still have significant drawbacks and are difficult to meet the needs of efficient large-scale culture: First, the movement mode is limited, only enabling the culture dish to revolve around the axis (oscillation angle ≤30°), and the culture dish cannot rotate on its own. This causes adipose tissue blocks to easily accumulate in local areas of the culture dish, resulting in a small contact area with digestive enzymes, leading to local over-digestion and insufficient digestion of the interior, and a low stem cell extraction rate. Second, the temperature control method is rudimentary, relying on air heating inside the incubator, resulting in large temperature fluctuations, which easily affect enzyme activity and further reduce cell release efficiency. Third, the culture scale is limited, with the device only having a single placement tank, resulting in a small culture volume per batch, which cannot meet the needs of industrial mass production.

[0005] Therefore, existing adipose stem cell exosome culture devices have significant shortcomings in terms of motion structure design, temperature control precision, and culture efficiency. There is an urgent need for a technical solution that can achieve compound motion, precise temperature control, and batch culture to solve industry pain points such as uneven contact between adipose tissue blocks and digestive enzymes, low cell extraction rate, and insufficient exosome yield. Summary of the Invention

[0006] The main technical problem to be solved by the present invention is to provide a simple overall structure for adipose stem cell exosome culture device and culture method, which can improve the temperature control accuracy during the culture of adipose stem cell exosomes, enable batch culture, and solve the problems of uneven contact between adipose tissue blocks and digestive enzymes and low cell extraction rate.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solution: A device for culturing adipose-derived stem cell exosomes includes a culture chamber containing two independent sealed enclosures: a water bath and a power control box. A first sliding frame is slidably mounted near the upper end of the water bath. Multiple evenly spaced mounting plates are fixedly mounted on the first sliding frame. Multiple evenly spaced closed culture dishes are rotatably mounted on the mounting plates, all of which are completely immersed in a constant-temperature bath medium. A temperature control device is installed on the inner wall of the water bath below the mounting plates to precisely stabilize the constant-temperature bath medium at a preset culture temperature and provide real-time temperature data feedback. A dual-shaft motor is fixedly mounted in the power control box away from the water bath. Both output shafts of the dual-shaft motor are connected to a multi-link device, the other end of which is fixedly connected to the first sliding frame, driving the first sliding frame to perform horizontal reciprocating sliding.

[0008] The following are further optimizations of the above technical solution by the present invention: The incubator includes a box body, which is divided into two independent sealed boxes, a water bath box and a power control box, by a middle partition plate. On the inner wall of the water bath box, a rack parallel to the length direction of each mounting plate is fixedly installed directly below each mounting plate.

[0009] Further optimization: Multiple evenly spaced drive shafts are rotatably mounted on the upper surface of the mounting plate via bearings. The drive shafts pass through the upper and lower end faces of the mounting plate, and gears are fixedly sleeved on the lower end of the drive shafts. All gears on each mounting plate are meshed with the corresponding rack below. The closed culture dish can be detachably installed on the upper end of the drive shaft.

[0010] Further optimization: The constant temperature device includes multiple electric heating rods fixedly installed on the middle partition of one side of the water bath, and multiple temperature sensors are also evenly installed inside the water bath.

[0011] Further optimization: A motor mounting platform is vertically arranged on the bottom surface of the power control box, away from the middle partition. The dual-output shaft motor is fixedly mounted on the top of the motor mounting platform with bolts.

[0012] Further optimization: The multi-link device includes a mounting frame fixedly installed in the power control box. Lower support bearings are fixedly installed on both sides of the bottom end of the mounting frame near the dual-output shaft motor. The dual output shaft ends of the dual-output shaft motor are connected to the multi-link group. The lower support bearings can provide a stable rotation fulcrum for the swing of the multi-link group.

[0013] Further optimization: The multi-link assembly includes a crank fixedly mounted on a single-side power output shaft of a dual-output-shaft motor. The end of the crank away from the motor output shaft is hinged to a rear connecting rod, and the other end of the rear connecting rod is hinged to one end of a rocker arm, forming a movable transmission link. The lower end of the rocker arm is rotatably mounted in the inner ring of the lower support bearing. The upper ends of the rocker arms on both sides are fixedly connected by a central connecting shaft. The upper end of the rocker arm is also hinged to a front connecting rod, and the end of the front connecting rod away from the rocker arm is hinged to a second sliding frame.

[0014] Further optimization: The mounting frame is symmetrically fixedly installed with U-shaped sliding grooves extending in the horizontal direction on both sides near the front connecting rod. Rubber wheels are rotatably installed on both sides of the second sliding frame at positions corresponding to the U-shaped sliding grooves. The rubber wheels and the U-shaped sliding grooves form a low-friction rolling engagement to ensure that the second sliding frame slides stably along the U-shaped sliding grooves.

[0015] Further optimization: Multiple evenly spaced connecting plates are vertically fixedly installed at one end of the first sliding frame near the middle partition, and the other end of the first sliding frame passes through the middle partition and is fixedly connected to the second sliding frame.

[0016] The present invention also provides a method for culturing adipose-derived stem cell exosomes, based on the above-mentioned adipose-derived stem cell exosome culture device, comprising the following steps: S1. After cleaning the collected adipose tissue, cut it into uniform tissue fragments, then mix it thoroughly with collagenase digestion solution in a certain proportion to ensure that the tissue fragments come into initial contact with the digestive enzyme. Aseptically aliquot the mixed adipose tissue sample into multiple closed culture dishes. S2. Add constant temperature bath medium into the water bath tank; S3. Close the lid and lock it using the snap-locking structure. Set parameters using the touch panel on the surface of the box. S4. Click the start button on the touch panel. The control system first starts the thermostat, controlling the electric heating rod to heat up quickly at maximum power. At the same time, multiple temperature sensors collect temperature data in real time and feed it back to the system. S5. When the temperature of the constant temperature bath medium in the water bath reaches the threshold, the control system adjusts the power of the electric heating rod to stabilize the temperature of the constant temperature bath medium at 37℃. S6. The control system starts the dual-output shaft motor. The dual-output shaft motor converts the rotational motion into the horizontal reciprocating sliding of the first sliding frame through a multi-link device at a preset speed. At the same time, it drives the mounting plate and the closed culture dish to slide horizontally back and forth. During the translation, the gear meshes with the rack and pinion, driving the transmission shaft and the closed culture dish to rotate regularly in both directions around its own central axis, so that the fat tissue block in the closed culture dish can come into full contact with the digestive enzymes. S7. During the cultivation process, the control system automatically monitors the operating status. If any abnormalities such as overheating or leakage occur, an alarm will be triggered immediately and corresponding safety protection operations will be executed. S8. After the culture is completed, the control system issues a termination command, the dual-shaft motor stops smoothly, the electric heating rod stops working, and at the same time the touch panel displays that the culture is complete and emits a prompt sound; S9. Release the constant temperature bath medium, open the lid, and remove the sealed petri dishes one by one to the ultra-clean workbench before proceeding to the next step. S10. Key data during the culture process can be exported via the touch panel, facilitating experimental traceability and enabling the cleaning and disinfection of various components inside the chamber to ensure a sterile environment for the next use.

[0017] The present invention adopts the above technical solution and has the following beneficial effects: This invention employs the aforementioned technical solution, which is ingeniously conceived and rationally structured. A dual-axis motor drives a multi-link device to achieve horizontal reciprocating sliding of the first sliding frame. Simultaneously, the meshing transmission between the gear below the mounting plate and the rack inside the water bath drives the closed culture dish to rotate regularly in both directions around its central axis. This design completely overcomes the limitations of traditional devices with their single-motion operation, allowing the adipose tissue mass to tumble within the culture dish and fully contact the collagenase digestion solution. This avoids problems of localized over-digestion or insufficient internal digestion, significantly improving the release efficiency and extraction purity of adipose stem cells, laying the foundation for high-yield exosome production in the future.

[0018] Simultaneously, a water bath heating method is employed, using multiple evenly distributed electric heating rods to achieve rapid multi-point heating. Multiple temperature sensors within the water bath collect temperature data in real time and feed it back to the control system. The control system stabilizes the temperature of the constant-temperature bath medium within a range of 37°C by adjusting the power of the electric heating rods. Compared to traditional air heating methods, this results in less temperature fluctuation, maximizing the maintenance of collagenase activity and the stability of adipose-derived stem cells, avoiding cell damage caused by temperature fluctuations, and ensuring the uniformity of culture quality.

[0019] In addition, multiple evenly spaced mounting plates are fixedly installed on the first sliding frame. Each mounting plate can carry multiple closed culture dishes through multiple drive shafts, realizing simultaneous culture of multiple samples and greatly increasing the scale of a single culture. At the same time, the device has a compact structural design, efficient transmission of each component, and the power output of the dual-output shaft motor can stably drive the load of multiple platforms for long-term operation. It not only meets the flexibility requirements of laboratory batch experiments, but also adapts to the large-scale requirements of industrial mass production, reducing the unit culture cost.

[0020] The core transmission components of this device (first sliding frame, slide rail, rack, gear, drive shaft, etc.) are made of POM (polyoxymethylene), with some key parts reinforced with 316L stainless steel. POM material has excellent water resistance and corrosion resistance, and does not swell or release harmful substances when immersed in constant temperature bath medium for a long time, completely avoiding debris contamination caused by metal corrosion. The closed culture dish adopts a double sealing structure of threaded engagement + medical-grade silicone sealing ring. A silicone sealing sleeve is set at the penetration between the connecting plate and the intermediate partition. The multiple sealing design effectively prevents leakage of constant temperature bath medium and intrusion of external contaminants, ensuring a sterile and clean culture environment, which meets the stringent requirements of stem cell exosome culture.

[0021] The multi-link device, through the coordinated transmission of cranks, connecting rods, and rocker arms, combined with the low-friction rolling contact of U-shaped slides and rubber wheels, ensures smooth movement of the sliding frame with low resistance and low wear. All components are integrally injection molded or bolted together, resulting in high structural strength and assembly precision, making them resistant to deformation or jamming during long-term operation. In addition, the bottom of the water bath is equipped with an outlet with a shut-off valve for easy media replacement and equipment cleaning. The touch panel supports the export and traceability of culture data. The overall design balances operational stability and ease of maintenance, extending the service life of the device while reducing subsequent operation and maintenance costs.

[0022] The present invention will be further described below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the overall structure in an embodiment of the present invention; Figure 2 This is a cross-sectional view of the overall structure in an embodiment of the present invention; Figure 3 This is a three-dimensional cross-sectional view of the overall structure in an embodiment of the present invention; Figure 4 This is a three-dimensional structural diagram of the multi-link device in cooperation with the first sliding frame in an embodiment of the present invention; Figure 5 This is a schematic diagram of the structure of the first sliding frame in an embodiment of the present invention; Figure 6 This is an embodiment of the present invention. Figure 3 Enlarged schematic diagram of the structure at point A in the middle.

[0024] In the diagram: 1. Incubator; 11. Chamber body; 110. Water bath; 111. Power control box; 112. Middle partition; 12. Cover; 2. Mounting plate; 21. First sliding frame; 211. Rack; 212. Gear; 213. Drive shaft; 214. Bearing; 22. Closed culture dish; 23. Connecting plate; 3. Multi-link device; 31. Mounting frame; 32. Motor mounting platform; 33. Multi-link assembly; 331. Crank; 332. Rear connecting rod; 333. Front connecting rod; 334. U-shaped slide; 335. Rubber wheel; 336. Rocker arm; 34. Lower support bearing; 35. Intermediate connecting shaft; 36. Second sliding frame; 4. Dual-shaft motor; 5. Temperature control device; 51. Electric heating rod; 52. Temperature sensor; 6. Touch panel. Detailed Implementation

[0025] like Figure 1-6 As shown: A fat stem cell exosome culture device includes a culture box 1. The culture box 1 contains two independent sealed boxes: a water bath 110 and a power control box 111. A first sliding frame 21 is slidably installed near the upper end of the water bath 110. Multiple evenly spaced mounting plates 2 are fixedly installed on the first sliding frame 21. Multiple evenly spaced closed culture dishes 22 are rotatably installed on the mounting plates 2. All closed culture dishes 22 are completely immersed in a constant temperature bath medium. A constant temperature device 5 is provided on the inner wall of the water bath 110 below the mounting plates 2 to accurately stabilize the constant temperature bath medium at a preset culture temperature and provide real-time temperature data feedback. A dual-shaft motor 4 is fixedly installed in the power control box 111 at a position away from the water bath 110. The dual-shaft ends of the dual-shaft motor 4 are driven by a multi-link device 3. The other end of the multi-link device 3 is fixedly connected to the first sliding frame 21, driving the first sliding frame 21 to perform horizontal reciprocating sliding.

[0026] The incubator 1 includes a box body 11, which is divided into two independent sealed boxes, a water bath box 110 and a power control box 111, by a middle partition plate 112 installed in the middle.

[0027] A flip-top lid 12 is hinged to one side of the box body 11. A medical-grade silicone sealing gasket is embedded in the inner edge of the lid 12, and a snap-locking structure is provided at the closure of the lid 12 and the box body 11. When the lid 12 is closed and locked, it can form a reliable seal with the box body 11, which can prevent external dust and moisture from entering the box and reduce the heat loss of the constant temperature bath medium in the water bath 110, thus ensuring the airtightness and constant temperature stability of the culture environment.

[0028] A touch panel 6 is also fixedly installed on one side of the housing 11.

[0029] In this embodiment, the temperature of the constant temperature bath medium is set to 37°C for water.

[0030] The first sliding frame 21 is made of multiple POM (polyoxymethylene) sheets of different widths, which are integrally injection molded to form a stable frame-type load-bearing structure. 316L stainless steel reinforcing ribs are added inside (arranged along the sliding direction). This not only utilizes the excellent water resistance and corrosion resistance of POM material to prevent rusting when immersed in constant temperature bath medium for a long time, but also improves the overall load-bearing strength and deformation resistance through the reinforcing ribs, ensuring the long-term stability of the device.

[0031] Near the upper end of the inner walls on both sides of the water bath 110, there are symmetrically fixed long strip slide rails that are adapted to the sliding trajectory of the first sliding frame 21. The slide rails are made of POM (polyoxymethylene) material and are integrally injection molded. They are arranged along the length of the water bath 110 and their surfaces are mirror polished to reduce sliding friction resistance.

[0032] Four pulleys (two on each side, located at the ends and middle of the first sliding frame 21) are rotatably mounted on both sides of the slide rail via corrosion-resistant sealed bearings. The pulleys are also made of POM material, forming a low-friction fit with the slide rail. Their contours precisely match the slide rail, resulting in low rolling resistance and no wear or abnormal noise. The POM material does not swell or release harmful substances when immersed in a 37°C constant temperature bath medium for a long time, fully meeting the biosafety requirements for cell culture.

[0033] In this embodiment, the corrosion-resistant sealed bearing is selected from the LDK brand SUC series, which is commercially available.

[0034] By combining the slide rail made of POM material, the first sliding frame 21 (including stainless steel reinforcing ribs) and pulleys, the risk of corrosion in the water bath environment is completely avoided. Through the matching transmission and structural reinforcement of the same material, and with the power output of the multi-link device 3, the first sliding frame 21 can slide smoothly back and forth along the preset trajectory in the water bath 110, ensuring that the closed culture dish 22 carried on it moves stably and without deviation.

[0035] Multiple mounting plates 2 are integrally injection molded from POM (polyoxymethylene) material, with 316L stainless steel reinforcing plates embedded inside. These plates are adapted to the contours of mounting plates 2 and are centrally positioned along the thickness direction. The stainless steel reinforcing plates compensate for the rigidity shortcomings of POM material, significantly improving the load-bearing strength and deformation resistance of mounting plates 2, and can stably support the weight of multiple closed culture dishes 22. Furthermore, the excellent water resistance, corrosion resistance, and rust-free properties of POM material, combined with the corrosion resistance of 316L stainless steel, completely prevent corrosion caused by long-term immersion in the constant temperature bath medium, prevent metal debris from falling off and contaminating the constant temperature bath medium and culture samples, and ensure the sterile and clean culture environment.

[0036] like Figure 5-6 As shown, on the inner wall of the water bath 110, directly below each mounting plate 2, there is a rack 211 that is parallel to the length direction of the mounting plate 2. The rack 211 is made of POM (polyoxymethylene) material and is injection molded in one piece. The tooth surface is finely ground to reduce meshing friction. Relying on the excellent water resistance and corrosion resistance of POM material, rust and pollution are completely avoided in the water bath environment.

[0037] The upper surface of the mounting plate 2 is rotatably mounted with multiple evenly spaced drive shafts 213 via bearings 214.

[0038] The drive shaft 213 is integrally formed from POM material, and its surface is polished to reduce rotational resistance.

[0039] The bearing 214 is a corrosion-resistant sealed bearing with an IP68 waterproof rating (compliant with GB / T4208-2017 standard). The outer ring is interference-fitted with the reserved mounting hole of the mounting plate 2. The drive shaft 213 passes through the upper and lower end faces of the mounting plate 2 and is fixedly connected to the inner ring of the bearing 214 to ensure smooth rotation and prevent water bath medium from seeping into the interior of the bearing 214.

[0040] The closed culture dish 22 can be detachably installed on the upper end of the drive shaft 213 through a threaded locking part made of POM material. After installation, the bottom of the culture dish fits tightly with the upper surface of the mounting plate 2. The POM material accessory has strong compatibility with the main structure and has no risk of rust, avoiding loosening and falling off during sliding or rotation.

[0041] The closed culture dish 22 is also equipped with a sealing cap, which is sealed by setting threads and a silicone sealing ring. The specific sealing structure is known in the prior art and will not be described in detail here.

[0042] The lower end of the drive shaft 213 is fixedly fitted with a gear 212. The gear 212 is integrally injection molded from POM material and is located on the same vertical plane as the rack 211. All gears 212 on each mounting plate 2 are meshed with the corresponding rack 211 below.

[0043] When the multi-link device 3 drives the first sliding frame 21 to move the mounting plate 2 horizontally back and forth, the gear 212 meshes with the rack 211, resulting in a low coefficient of friction and excellent wear resistance. This drives the transmission shaft 213 and the closed culture dish 22 to rotate regularly in both directions around their own central axis, ensuring a clean culture environment with no risk of rust or contamination throughout the process.

[0044] In this embodiment, the closed culture dish 22 adopts a sealing structure design adapted to water bath oscillation scenarios: a medical-grade silicone sealing ring is embedded between the dish body and the culture dish lid, and the culture dish lid and dish body are locked by screwing together to form a reliable water seal effect, which can effectively prevent the constant temperature bath medium from seeping into the dish or the culture solution from leaking, thus avoiding contamination of the culture sample. This sealing method is based on the mature sealing principle of existing water bath culture and optimized for adaptation. Its specific assembly details are common knowledge to those skilled in the art and will not be described in detail here.

[0045] like Figure 2-3 As shown, the constant temperature device 5 includes multiple electric heating rods 51 fixedly installed on a partition plate 112 on one side inside the water bath 110.

[0046] The electric heating rods 51 are evenly distributed along the height of the partition and extend to the core water area inside the water bath 110. By heating at multiple points evenly, the heating rate is increased, ensuring that the temperature of the constant temperature bath medium quickly becomes uniform.

[0047] The water bath 110 is also equipped with multiple temperature sensors 52 evenly installed. The probes of the multiple temperature sensors 52 are distributed in different areas of the water bath 110, which can comprehensively collect the temperature data of the constant temperature bath medium and provide reliable feedback for precise temperature control.

[0048] The electric heating rod 51 adopts a corrosion-resistant and sealed design. Its heating tube shell is made of 316L stainless steel, and the end interface is equipped with a waterproof sealing sleeve to prevent water bath medium from seeping into the interior and causing short circuits or component damage. The temperature sensor 52's probe is encapsulated with medical-grade materials, and the cable connection is sealed. Neither of them releases harmful substances and is resistant to water bath medium corrosion. Long-term immersion will not produce rust, debris, or other contaminants, ensuring a clean and sterile culture environment while maintaining its own structural integrity and functional stability.

[0049] A motor mounting platform 32 is vertically arranged on the bottom surface of the power control box 111, away from the middle partition 112. The dual-output shaft motor 4 is fixedly mounted on the top of the motor mounting platform 32 by bolts.

[0050] In this embodiment, the dual-output shaft motor 4 is a 57BYG250H type stepper dual-output shaft motor, which can be obtained commercially. The dual-output shaft motor 4 transmits torque through the multi-link device 3 on both sides of the dual output shaft, converting its own rotational motion into the horizontal reciprocating sliding of the first sliding frame 21, which in turn drives the closed culture dish 22 to move horizontally back and forth, so that the fat tissue block and digestive enzyme in the culture dish generate sufficient relative motion, avoiding local mixing dead zones.

[0051] like Figure 4 As shown, the multi-link device 3 includes a mounting frame 31 fixedly installed in the power control box 111, and lower support bearings 34 are fixedly installed on both sides of the bottom end of the mounting frame 31 near the dual output shaft motor 4.

[0052] The dual-output shaft motor 4 has multiple linkages 33 connected to both output shaft ends. The symmetrical transmission design ensures balanced power output on both sides and avoids sliding deviation of the first sliding frame 21.

[0053] The lower support bearing 34 provides a stable rotational fulcrum for the swing of the multi-link assembly 33.

[0054] The multi-link assembly 33 includes a crank 331 fixedly mounted on the power output shaft of a dual-output-shaft motor 4 on one side. The end of the crank 331 away from the motor output shaft is hinged to a rear connecting rod 332. The other end of the rear connecting rod 332 is hinged to one end of a rocker arm 336, forming a movable transmission link.

[0055] The lower end of the rocker arm 336 is rotatably mounted in the inner ring of the lower support bearing 34 via a rotating shaft. The lower support bearing 34 provides a stable and low-resistance rotation fulcrum for the reciprocating swing of the rocker arm 336.

[0056] The upper ends of the rocker arms 336 on both sides are fixedly connected by the intermediate connecting shaft 35 to ensure that the rocker arms 336 on both sides swing synchronously and ensure balanced power transmission.

[0057] The upper end of the rocker arm 336 is also hinged to a front connecting rod 333, and the end of the front connecting rod 333 away from the rocker arm 336 is hinged to a second sliding frame 36.

[0058] The mounting frame 31 is symmetrically fixedly mounted with U-shaped slide grooves 334 extending in the horizontal direction on both sides near the front connecting rod 333. Rubber wheels 335 are rotatably mounted on both sides of the second sliding frame 36 at positions corresponding to the U-shaped slide grooves 334.

[0059] The rubber wheel 335 and the U-shaped slide groove 334 form a low-friction rolling fit to ensure that the second sliding frame 36 slides stably along the U-shaped slide groove 334.

[0060] Multiple evenly spaced connecting plates 23 are vertically fixedly installed at one end of the first sliding frame 21 near the middle partition 112. The connecting plates 23 are made of POM material, and the end away from the first sliding frame 21 passes through the reserved through hole on the middle partition 112 and is fixedly connected to the second sliding frame 36.

[0061] The connection between the connecting plate 23 and the intermediate partition plate 112 is fitted with a medical-grade silicone sealing sleeve, forming a double sealing structure, which effectively prevents the constant temperature bath medium in the water bath 110 from leaking into the power control box 111.

[0062] With this design, after the dual-output shaft motor 4 is started, the dual output shafts of the dual-output shaft motor 4 drive the cranks 331 on both sides to rotate synchronously. The cranks 331 pull the connecting rod 332 through the hinge point to make a push-pull motion, which in turn drives the rocker arm 336 to swing back and forth around the rotating shaft of the lower support bearing 34. The rocker arms 336 on both sides maintain synchronous movement through the intermediate connecting shaft 35. The upper end of the rocker arm 336 converts the swing motion into the horizontal reciprocating sliding of the second sliding frame 36 through the front connecting rod 333. At this time, the rubber wheels 335 on both sides of the second sliding frame 36 roll smoothly along the U-shaped sliding groove 334, reducing motion resistance and wear.

[0063] The second sliding frame 36 drives the first sliding frame 21 to move horizontally and reciprocally in sync through the connecting plate 23, thereby driving the mounting plate 2 and the closed culture dish 22 to move along the slide rail inside the water bath 110; at the same time, the gear 212 below the mounting plate 2 meshes with the rack 211 fixed inside the water bath 110 during the translation of the mounting plate 2, driving the transmission shaft 213 and the closed culture dish 22 to rotate regularly in both directions around their own central axis.

[0064] Ultimately, the power is transmitted through the multi-link assembly 33, enabling the closed culture dish 22 to simultaneously complete the combined motion of horizontal reciprocating sliding and its own rotation. This ensures that the internal adipose tissue blocks and digestive enzymes are fully mixed without any dead corners, thereby improving the efficiency of stem cell release and the uniformity of culture.

[0065] The power control box 111 is also equipped with a control system for controlling the operation of the culture device. The control system is based on a microcontroller, and the dual-output shaft motor 4 is electrically connected to the control system and receives speed adjustment and start / stop control signals.

[0066] Thermostatic control components: The multiple electric heating rods 51 and multiple temperature sensors 52 in the thermostatic device 5 are all connected to the control system signal. The temperature sensors 52 upload temperature data in real time, and the electric heating rods 51 receive power adjustment commands.

[0067] Human-machine interface component: The touch panel 6 adopts a capacitive touch screen; the touch panel 6 communicates bidirectionally with the control system, supports user input of parameters, viewing of operating status, retrieval of historical data, and receiving alarm information from the system. The operation interface adopts a graphical design, including four functional areas: parameter setting, operation monitoring, alarm recording, and data export, which are intuitive and easy to understand.

[0068] The specific control principle of the control system is well known in the prior art and will not be elaborated here.

[0069] The bottom of the water bath 110 has an integrally formed liquid outlet, which is equipped with a POM material shut-off valve. The inner wall of the liquid outlet is machined with internal threads, which can be connected to an external guide hose. By opening and closing the shut-off valve, the constant temperature bath medium in the water bath 110 can be quickly and thoroughly discharged, which facilitates subsequent equipment cleaning, maintenance and medium replacement, and effectively avoids the risk of pollution caused by medium residue.

[0070] The present invention also provides a method for culturing adipose-derived stem cell exosomes, based on the above-mentioned adipose-derived stem cell exosome culture device, comprising the following steps: S1. After cleaning the collected adipose tissue, cut it into uniform tissue fragments and mix it thoroughly with collagenase digestion solution in a certain proportion to ensure that the tissue fragments come into initial contact with the digestive enzyme. Aseptically aliquot the mixed adipose tissue sample into multiple closed culture dishes 22 and seal them with the screw caps and silicone sealing rings to form a reliable water seal, preventing media leakage or sample contamination during subsequent culture. Next, the sealed petri dish 22 is installed sequentially on the upper end of the drive shaft 213 using threaded locking, ensuring that the installation is secure and without looseness; S2. Add constant temperature bath medium to the water bath 110. In this embodiment, liquid water is selected as the constant temperature bath medium to ensure that the liquid surface of the constant temperature bath medium can completely submerge the closed culture dish 22. S3. Close the box lid 12 and lock it with a snap-locking structure. Set the parameters through the touch panel 6 on the surface of the box body 11. Set the target culture temperature to 37℃ and the culture time to 1-1.5 hours. Set the oscillation frequency by the rotation speed of the dual-output shaft motor 4. That is, for every rotation of the dual output shaft of the dual output shaft motor 4, the multi-link device 3 drives the first sliding frame 21 and the second sliding frame 36 to complete one reciprocating slide (i.e., one oscillation). Set the oscillation frequency to 20 times / minute. At the same time, the rotation speed of the closed culture dish 22 is 8 revolutions / minute. S4. Click the start button on the touch panel 6. The control system first starts the thermostat 5, controls the electric heating rod 51 to heat up quickly at maximum power, and at the same time, multiple temperature sensors 52 collect temperature data in real time and feed it back to the system; S5. When the temperature of the constant temperature bath medium in the water bath 110 reaches the threshold, the control system adjusts the power of the electric heating rod 51 to stabilize the temperature of the constant temperature bath medium at 37℃. S6. The control system starts the dual-output shaft motor 4. The dual-output shaft motor 4 converts the rotational motion into the horizontal reciprocating sliding of the first sliding frame 21 through the multi-link device 3 at a preset speed. At the same time, it drives the mounting plate 2 and the closed culture dish 22 to slide horizontally back and forth. During the translation, the gear 212 meshes with the rack 211 to drive the transmission shaft 213 and the closed culture dish 22 to rotate regularly in both directions around its own central axis, so that the fat tissue block in the closed culture dish 22 can fully contact the digestive enzymes. S7. During the cultivation process, the control system automatically monitors the operating status. If any abnormalities such as overheating or leakage occur, an alarm will be triggered immediately and corresponding safety protection operations will be executed. S8. After the culture is completed, the control system issues a termination command, the dual-shaft motor 4 stops smoothly, the electric heating rod 51 stops working, and at the same time the touch panel 6 displays that the culture is complete and emits a prompt sound; S9. Open the shut-off valve to release the constant temperature bath medium, open the box cover 12, and take out the closed culture dishes 22 one by one and transfer them to the ultra-clean workbench, and then proceed to the next step of processing; S10. Key data during the culture process can be exported via the touch panel 6, facilitating experimental traceability. All components inside the chamber 11 can be cleaned and disinfected to ensure a sterile environment for the next use.

[0071] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. An apparatus for culturing adipose-derived stem cell exosomes, comprising an incubator (1), characterized in that... The incubator (1) contains two independent sealed boxes: a water bath (110) and a power control box (111). A first sliding frame (21) is slidably installed near the upper end of the water bath (110). Multiple evenly spaced mounting plates (2) are fixedly installed on the first sliding frame (21). Multiple evenly spaced closed culture dishes (22) are rotatably installed on the mounting plates (2). All closed culture dishes (22) are completely immersed in the constant temperature bath medium. The water bath (110)... A constant temperature device (5) is installed on the inner wall below the mounting plate (2) to precisely stabilize the constant temperature bath medium at the preset culture temperature and provide real-time temperature data feedback; a dual-output shaft motor (4) is fixedly installed in the power control box (111) at a position away from the water bath (110). The dual output shaft ends of the dual-output shaft motor (4) are both connected to a multi-link device (3). The other end of the multi-link device (3) is fixedly connected to the first sliding frame (21) to drive the first sliding frame (21) to perform horizontal reciprocating sliding.

2. The adipose-derived stem cell exosome culture device according to claim 1, characterized in that... The incubator (1) includes a box body (11), which is divided into two independent sealed boxes, a water bath box (110) and a power control box (111), by a middle partition plate (112) installed in the middle. A rack (211) parallel to the length direction of the mounting plate (2) is fixedly installed on the inner wall of the water bath box (110) at a position directly below each mounting plate (2).

3. The adipose-derived stem cell exosome culture device according to claim 2, characterized in that... The mounting plate (2) has multiple evenly spaced drive shafts (213) rotatably mounted on its upper surface. The drive shafts (213) pass through the upper and lower end faces of the mounting plate (2). The lower end of the drive shafts (213) is fixedly fitted with gears (212). All gears (212) on each mounting plate (2) are meshed with the corresponding racks (211) below. The closed culture dish (22) is detachably mounted on the upper end of the drive shaft (213).

4. The adipose-derived stem cell exosome culture device according to claim 3, characterized in that... The constant temperature device (5) includes multiple electric heating rods (51) fixedly installed on a partition plate (112) on one side inside the water bath (110). Multiple temperature sensors (52) are also evenly installed inside the water bath (110).

5. The adipose-derived stem cell exosome culture device according to claim 4, characterized in that... The power control box (111) has a motor mounting platform (32) vertically arranged on the bottom surface away from the middle partition (112), and the dual-output shaft motor (4) is fixedly installed on the top of the motor mounting platform (32).

6. The adipose-derived stem cell exosome culture device according to claim 5, characterized in that... The multi-link device (3) includes a mounting frame (31) fixedly installed in the power control box (111). The bottom end of the mounting frame (31) is fixedly installed with lower support bearings (34) on both sides near the dual-output shaft motor (4). The dual output shaft ends of the dual-output shaft motor (4) are connected to the multi-link group (33). The lower support bearings (34) can provide a stable rotation fulcrum for the swing of the multi-link group (33).

7. The adipose-derived stem cell exosome culture device according to claim 6, characterized in that... The multi-link assembly (33) includes a crank (331) fixedly mounted on the single-side power output shaft of a dual-output-shaft motor (4). A rear connecting rod (332) is hinged to one end of the crank (331) away from the motor output shaft. The other end of the rear connecting rod (332) is hinged to one end of a rocker arm (336) to form a movable transmission link. The lower end of the rocker arm (336) is rotatably mounted in the inner ring of a lower support bearing (34). The upper ends of the rocker arms (336) on both sides are fixedly connected by the intermediate connecting shaft (35). The upper end of the rocker arm (336) is also hinged to the front connecting rod (333). The end of the front connecting rod (333) away from the rocker arm (336) is hinged to the second sliding frame (36).

8. The adipose-derived stem cell exosome culture device according to claim 7, characterized in that... The mounting frame (31) is symmetrically fixed with U-shaped grooves (334) extending horizontally on both sides near the front connecting rod (333). Rubber wheels (335) are rotatably installed on both sides of the second sliding frame (36) at positions corresponding to the U-shaped grooves (334). The rubber wheels (335) and the U-shaped grooves (334) form a low-friction rolling fit to ensure that the second sliding frame (36) slides stably along the U-shaped grooves (334).

9. The adipose-derived stem cell exosome culture device according to claim 8, characterized in that... The first sliding frame (21) has a number of evenly spaced connecting plates (23) vertically fixedly installed at one end near the middle partition plate (112), and the end away from the first sliding frame (21) passes through the middle partition plate (112) and is fixedly connected to the second sliding frame (36).

10. A method for culturing adipose-derived stem cell exosomes, based on the adipose-derived stem cell exosome culture device according to claim 9, characterized in that... Includes the following steps: S1. After cleaning the collected adipose tissue, cut it into uniform tissue fragments, and then mix it thoroughly with collagenase digestion solution in proportion to ensure that the tissue fragments come into initial contact with the digestive enzyme. Aseptically dispense the mixed adipose tissue sample into multiple closed culture dishes (22). S2. Add constant temperature bath medium into the water bath tank (110); S3. Close the lid (12) and lock it with a snap-locking structure. Set the parameters using the touch panel (6) on the surface of the box (11). S4. Click the start button on the touch panel (6). The control system first starts the constant temperature device (5), controls the electric heating rod (51) to heat up quickly at maximum power, and at the same time, multiple temperature sensors (52) collect temperature data in real time and feed it back to the system; S5. When the temperature of the constant temperature bath medium in the water bath (110) reaches the threshold, the control system adjusts the power of the electric heating rod to stabilize the temperature of the constant temperature bath medium at 37℃. S6. The control system starts the dual-output shaft motor (4). The dual-output shaft motor (4) converts the rotational motion into the horizontal reciprocating sliding of the first sliding frame (21) through the multi-link device (3) at the preset speed. At the same time, it drives the mounting plate (2) and the closed culture dish (22) to slide horizontally back and forth. During the translation process, the gear (212) meshes with the rack (211) to drive the transmission shaft (213) and the closed culture dish (22) to rotate regularly in both directions around their own central axis, so that the fat tissue block in the closed culture dish (22) can fully contact the digestive enzymes. S7. During the cultivation process, the control system automatically monitors the operating status. If any abnormalities such as overheating or leakage occur, an alarm will be triggered immediately and corresponding safety protection operations will be executed. S8. After the culture is completed, the control system issues an end command, the dual-shaft motor (4) stops smoothly, the electric heating rod (51) stops working, and at the same time the touch panel (6) displays that the culture is complete and emits a prompt sound; S9. Release the constant temperature bath medium, open the box cover (12), take out the closed culture dishes (22) one by one and transfer them to the ultra-clean workbench, and then proceed to the next step of processing; S10. Key data during the culture process can be exported via the touch panel (6) to facilitate experimental traceability. All components inside the chamber (11) can be cleaned and disinfected to ensure a sterile environment for the next use.

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