Gradient centrifugation leukocyte extraction equipment and method for inhibiting release of inflammatory factors

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to gradient centrifugation leukocyte extraction equipment and method for inhibiting release of inflammatory factors, the equipment comprises a centrifugal cavity with a cover body, a placing cavity is arranged in the cavity body, the placing cavity is equipped with an illuminating lamp, an image sensor, a temperature sensor, a cooling assembly and a heating assembly, and the bottom is connected with a centrifugal driving assembly; the supporting frame at the top end of the placement cavity is matched with the supporting block, and the equipment stability and the sample treatment effect are improved by combining microfiltration membranes and other structures. According to the extraction method, the equipment is utilized, through the four steps of equipment pre-adjustment, sample preparation and placement, centrifugal process synchronous adjustment (including photo-biological adjustment, precise temperature control, gas signal molecule intervention and dynamic monitoring feedback) and leukocyte extraction, inflammatory factor release is effectively inhibited while gradient centrifugal separation is performed, the leukocyte extraction quality and activity are improved, and the leukocyte extraction efficiency is improved. The method is suitable for efficient extraction and subsequent research application of leukocytes in clinical blood samples.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, specifically a gradient centrifugal white blood cell extraction device and method for inhibiting the release of inflammatory factors. BACKGROUND

[0002] White blood cell extraction technology, as a fundamental and critical cell separation method in modern biomedical field, is self-evident in its importance. From early diagnosis of diseases, disease monitoring, to in-depth study of immunological mechanisms, and even development of personalized cell therapy products and preparation of high-quality cosmetic active ingredients, efficient, high-purity and functional stable white blood cell acquisition is the prerequisite for the smooth development of many downstream applications. With the vigorous development of frontier biological technologies such as precision medicine and cell therapy, the requirement for the refinement of white blood cell extraction is increasing, especially the maintenance of cell physiological state during the extraction process and the protection of subsequent functional integrity, which has become a key indicator to measure the advantages and disadvantages of an extraction technology. After searching, a white blood cell extraction device with publication number CN110577884B was found, which was published on June 14, 2022. This patent provides a device that can automatically, quickly and stably extract white blood cells with high activity from undiluted blood, and can effectively remove lysed red blood cells. However, this technical solution mainly focuses on the extraction efficiency and activity maintenance of white blood cells, and does not involve the inhibition mechanism of inflammatory factor release. In addition, the device structure is complex, including multiple modules (such as extraction module, lysis module, etc.), which may increase the operation difficulty and equipment cost, and is not conducive to large-scale popularization and application.

[0003] After searching, a high-purity white blood cell extraction method and device with publication number CN108342357B was found, which was published on April 5, 2024. This patent realizes the efficient separation of white blood cells through static stratification and gradient centrifugation, and the purity of the extracted white blood cells can reach more than 97%, with high yield. However, this technical solution, although excellent in white blood cell purity and yield, does not consider how to inhibit the release of inflammatory factors during the extraction process, which may affect the functional integrity of the extracted white blood cells. At the same time, this method relies on specific separation bags and support bodies, and the precise control of centrifugation parameters is required, which may increase the operation complexity and process difficulty. The core design of the existing technology focuses on physical separation efficiency, without considering cell stress physiology and inflammatory regulation mechanism in the core design, which forms a non-obvious deep contradiction: even if the separation efficiency and purity are high, if the release of inflammatory factors during cell extraction cannot be effectively inhibited, the obtained cell sample may deviate from the original state in function, thereby affecting its ultimate application value and data reliability. Therefore, the present application provides a gradient centrifugal white blood cell extraction device and method for inhibiting the release of inflammatory factors. SUMMARY

[0004] To make up for the deficiencies of the prior art, at least one technical problem raised in the background art is solved.

[0005] The technical scheme adopted by the present application to solve its technical problems is: the gradient centrifugation white blood cell extraction device for inhibiting the release of inflammatory factors, comprising a centrifuge body, which constitutes the overall bearing structure of the device and is designed to provide a stable operating platform and define a relatively closed internal space. The side wall of the centrifuge body is rotatably connected to the cover body through a pivot bearing, and the cover body can perform a closing operation during the operation of the device to ensure the formation of a physically isolated and environmentally stable internal centrifugation chamber, thereby effectively isolating external environmental factors and maintaining the constancy of internal environmental parameters. The inner side of the centrifuge body is uniformly provided with at least one set of placement cavities along its circumference. The internal structure of the placement cavity is precisely designed to accurately accommodate and reliably fix a conical or cylindrical centrifugation container, thereby providing the necessary structural support and spatial positioning for the accurate position and stable state of the sample in the gradient centrifugation operation.

[0006] Further, the side wall of the placement cavity is symmetrically and equally provided with at least two mounting slots, which are precisely adapted in size and shape to the external contour of the specific component to be installed, to ensure the stable embedding and accurate alignment of the component. The inner side wall of the mounting slot is fixedly connected with a lighting lamp, which is configured as a light-emitting diode (LED) array with multi-spectrum emission capability, capable of accurately emitting monochromatic light of a specific wavelength. The adjustable range of the specific wavelength covers 540 nanometers to 630 nanometers, wherein the 540 nanometer wavelength light is suitable for non-invasive monitoring and light regulation of cells due to its weak absorption characteristics of hemoglobin; the 630 nanometer wavelength light can induce the regulation of mitochondrial function activity in cells due to its high-efficiency absorption characteristics of Cytochrome c Oxidase (CCO), thereby inducing the release of endogenous nitric oxide (NO) in cells and regulating the production of reactive oxygen species (ROS), and thus playing a role in inhibiting cell stress response and release of inflammatory factors. The output light power density of the lighting lamp can be dynamically adjusted according to the preset centrifugation stage and cell state, and the typical light power density range is 10 mW / cm² to 50 mW / cm², and the light illumination duration is accurately synchronized with the centrifugation program.

[0007] The image sensor is electrically connected to the illuminating lamp through an electrical signal cable, and is a high-resolution global shutter CMOS image sensor with a pixel array of 2560x1920 pixels, a frame rate of 60 frames per second, a spectral response range covering 400 nanometers to 950 nanometers, and excellent low-noise performance. The image sensor is used to capture image information of the liquid layering interface, cell layer sedimentation process and cell aggregation state inside the centrifuge container in real time, and convert it into high-fidelity digital electrical signals. The image data is analyzed in real time by an embedded image processing unit, realizing non-invasive dynamic evaluation of the white blood cell separation efficiency, layering clarity and potential stress indicators (such as cell morphological abnormalities and aggregation degree).

[0008] The temperature sensor is electrically connected to the control board through an electrical interface, and is used to monitor the temperature data of the internal environment of the placement cavity in real time, and accurately convert it into a digital electrical signal. The inner side wall of the placement cavity is symmetrically and stably fixed with a cooling assembly and a heating assembly, both of which are electrically connected to the temperature sensor through independent control circuits. The cooling assembly is a multistage thermoelectric cooler array based on the semiconductor Peltier effect, with a single-stage cooling capacity of 80W, and realizes accurate refrigeration through direct current voltage driving; the heating assembly is a ceramic PTC heating element with fast response characteristics, with a maximum heating power of 100W. The cooling assembly and the heating assembly work cooperatively with an integrated high-efficiency heat dissipation structure and a micro circulating fan, according to the temperature signal fed back by the temperature sensor in real time, and combined with a preset temperature control algorithm (such as PID control algorithm), to accurately and dynamically adjust the temperature inside the placement cavity, so as to maintain the temperature inside the placement cavity continuously and stably in a preset physiological suitable range, which can be set as 4℃ to 10℃, to minimize the metabolic activity of white blood cells and inhibit stress response.

[0009] The side wall of the centrifuge body is fixedly connected with a control board inside, which adopts a high-performance embedded microcontroller unit (MCU) platform, integrates a high-speed digital signal processor (DSP) and a programmable logic gate array (FPGA), and has a rich peripheral interface. The control board is electrically connected with the image sensor and the temperature sensor through a high-speed data bus and a special communication protocol. The control board is internally integrated with a data processing module, a control logic module and a human-computer interaction module. The data processing module is responsible for receiving and processing high-speed image data streams from the image sensor and accurate temperature data from the temperature sensor. The control logic module performs real-time analysis and decision-making on the data according to the preset gradient centrifugation program, light biological regulation program, temperature control program and gas signal molecule release program, and outputs accurate control instructions. The brightness and wavelength of the illumination lamp, the power of the cooling assembly and the heating assembly, and the gas flow rate and duration of the subsequent gas delivery path are accurately controlled through a digital analog converter (DAC) or a pulse width modulation (PWM) signal. The control board is also integrated with a fault diagnosis and safety protection module to ensure safe shutdown of the equipment in abnormal conditions. The side wall of the control board is provided with an adjustment button in the form of a high-precision rotary encoder, which has a press confirmation function, and is used by the operator to set and adjust the operating parameters of the control board, including centrifugal speed, centrifugal time, target temperature, light parameters (wavelength, intensity, duration) and gas molecule release parameters (gas type, concentration, release time), so as to realize fine and programmable control of the whole white blood cell extraction process environment parameters.

[0010] The side wall inside the cover is firmly connected with a gas storage box, which adopts high-pressure sealed design and is filled with inert carrier gas (such as high-purity nitrogen) and a specific proportion of gas signal molecule mixture. The gas signal molecule can include but is not limited to nitric oxide (NO), hydrogen sulfide (H2S) or carbon monoxide (CO), among which nitric oxide is preferred due to its anti-inflammatory, anti-apoptotic and vasodilatory effects on leukocytes at physiological concentrations, and its premixed concentration can be adjusted between 5 ppm and 200 ppm. The gas storage box is connected to the input end of the gas guide pump through a standard pneumatic connector. The inner side of the cover is structured to have a fixed cavity, which is designed in terms of geometry and size to accurately fix the sub-components inside, ensuring the structural stability during high-speed rotation of the centrifuge. The side wall of the fixed cavity is connected with a support rod made of high-strength lightweight alloy material and optimized in structure for providing reliable mechanical support for the gas guide pump, gas guide tube and related sensors, and buffering vibration. The side wall of the support rod is connected with a gas guide pump, which is a precision micro-step motor driven diaphragm pump with a flow rate that can be accurately adjusted from 0.1 mL / min to 10 mL / min with an adjustment accuracy of ±0.05 mL / min, and a built-in micro pressure sensor for real-time monitoring of gas output pressure to ensure the stability and accuracy of gas delivery. The input end of the gas guide pump is connected to the internal gas outlet end of the gas storage box through a pressure-resistant hose, ensuring effective extraction of the gas signal molecule. The output end of the gas guide pump is connected to at least one set of gas guide tubes through a micro pneumatic connector. The gas guide tube is made of polytetrafluoroethylene (PTFE) material with excellent biocompatibility, with an inner diameter of 0.5 mm, excellent chemical stability and low gas adsorption. The end of the gas guide tube is uniformly distributed and connected with a plurality of microporous gas injection holes with a diameter of 0.1 mm to 0.3 mm, which are arrayed to realize uniform dispersion of the gas signal molecule in the centrifugal chamber inside the centrifuge body, thereby ensuring that the leukocytes can be fully and uniformly exposed to the anti-inflammatory active gas signal molecule environment during centrifugal separation. The gas guide pump, the gas guide tube and the gas injection hole together constitute a precise gas delivery and dispersion path for accurately releasing the gas signal molecule stored in the gas storage box into the centrifugal environment inside the centrifuge body with a preset flow rate, duration and concentration, and realizing effective inhibition of leukocyte stress response through gas phase diffusion and dissolution in the medium.

[0011] As a preferred embodiment of the present application, the top end of the placement cavity is fixed with a support frame through a precision machining process, the support frame is made of medical-grade stainless steel material, has good corrosion resistance and mechanical strength. The top end of the support frame is provided with a detachable support block, the external contour of the support block is accurately matched with the internal geometry of the support frame, and the quick installation and disassembly are realized through the buckle or threaded connection. The side wall of the support block is fixed with a microfiltration membrane, the microfiltration membrane is made of polyether sulfone (PES) or polyvinylidene fluoride (PVDF) membrane material with a pore size of 0.2 microns to 5 microns, has good hydrophilicity, high porosity and flow rate, is used for further physical filtration of potential cell debris, impurities or incompletely lysed red blood cells in the process of white blood cell extraction, so as to improve the purity of the final white blood cell product and reduce the influence on subsequent analysis or therapeutic application. The side wall of the support frame is provided with an annular positioning groove along the inner periphery, the size and shape of the positioning groove are used to accurately accommodate and fix the specific components. The inside of the side wall of the positioning groove is fixed with a first magnetic ring, the first magnetic ring is made of high-performance neodymium-iron-boron permanent magnet material, and the magnetic field strength reaches 1.2 tesla. The bottom end of the support block is fixed with a second magnetic ring corresponding to the position of the first magnetic ring, the second magnetic ring is also made of high-performance neodymium-iron-boron permanent magnet material, and the polarity of the second magnetic ring and the polarity of the first magnetic ring are specially designed, so that a stable and quickly separable magnetic connection is formed between the second magnetic ring and the first magnetic ring, thereby realizing the quick, tool-free disassembly between the support block (together with the microfiltration membrane) and the support frame, facilitating the replacement or cleaning of the filter membrane. The top end of the support block is symmetrically and uniformly fixed with a plurality of high-elasticity rubber blocks, the rubber blocks are made of medical-grade silicone rubber material, have excellent shock absorption and buffering performance, and are designed to effectively absorb and attenuate mechanical stress transmitted by the imbalance of the centrifugal rotor or external vibration during centrifugation, thereby further reducing the shear stress borne by the white blood cells and minimizing cell damage and stress activation. The rubber block also has a sealing function to ensure the airtightness of the chamber.

[0012] The present application also provides a gradient centrifugation white blood cell extraction method for inhibiting the release of inflammatory factors, which is realized by using the above-mentioned device, and the specific steps include: First, the internal environmental temperature of the placement cavity of the gradient centrifugation white blood cell extraction device is pre-adjusted before the device is operated. According to the preset program, the control board instructs the cooling assembly and the heating assembly to work cooperatively, accurately controls and maintains the temperature in the placement cavity in the physiological low temperature range of 4°C to 10°C, so as to reduce the basic metabolic rate of cells and provide a low-stress initial environment for the subsequent centrifugal separation process.

[0013] Secondly, the blood sample to be separated and the pre-prepared gradient centrifugation liquid (e.g., lymphocyte separation medium) are carefully injected into the centrifuge container in a specific ratio. The gradient centrifugation liquid forms a stable density gradient layer in the centrifuge container according to its density difference. Then, the filled centrifuge container is stably placed in the preset position inside the device placement cavity. The device starts and executes the pre-programmed gradient centrifugation program, which includes multiple stages, each stage setting specific centrifugal speed, centrifugal time, and temperature control parameters.

[0014] After the centrifugation program starts, the method performs the following synchronous regulation strategy at specific stages of centrifugal separation: First, implement photobiomodulation: the control board accurately controls the light array according to the progress of the centrifugation program and real-time cell state feedback, making it emit monochromatic light of a specific wavelength, which can be 630 nanometers or 540 nanometers, at a set light power density (e.g., 20 mW / cm²) to continuously irradiate the white blood cell suspension inside the centrifuge container. The 630 nanometer wavelength light promotes the synthesis of adenosine triphosphate (ATP) by activating cytochrome c oxidase in white blood cell mitochondria and regulates reactive oxygen species (ROS) levels, thereby stabilizing cell membrane structure and inhibiting the activation of inflammatory signaling pathways such as NF-κB, significantly reducing the early release of inflammatory factors (such as TNF-α, IL-6). The duration of light irradiation can be dynamically adjusted according to the centrifugation stage, with a typical duration of 10 minutes to 30 minutes to ensure the full realization of photobiomodulation effects.

[0015] Second, maintain precise temperature control: the temperature sensor continuously monitors the temperature inside the placement cavity in real time. After receiving the temperature data, the control board accurately regulates the cooling or heating power of the cooling and heating components through the built-in PID control algorithm, ensuring that the white blood cells inside the centrifuge container are always at the preset, strictly constant physiological suitable temperature (e.g., 6℃±0.5℃). This constant low-temperature environment can effectively inhibit excessive metabolism and stress response of cells under centrifugal shear force, further reducing the production and release of inflammatory mediators.

[0016] Third, the introduction of gas signal molecules intervention: the control board in the specific critical stage of gradient centrifugation process (for example, the initial stage of cell sedimentation, the formation of stratification or extraction preparation period), instruct the gas pump to start. The gas pump extracts and transports the gas signal molecule mixture from the gas storage box at a preset accurate flow rate (for example, 5 mL / min), through the gas guide pipe and the gas injection hole array, uniformly disperses the gas signal molecule (for example, nitric oxide) into the air environment of the centrifugal chamber, and penetrates and dissolves in the centrifugal liquid. The nitric oxide activates the intracellular soluble guanylate cyclase (sGC), increases the level of cyclic guanosine monophosphate (cGMP), and then activates the downstream signaling pathway, plays its anti-inflammatory, anti-apoptotic, antioxidant and inhibits leukocyte adhesion and activation, thereby inhibiting the release of leukocyte stress inflammatory factors from the molecular level. The release duration of gas signal molecules is usually 5 to 15 minutes, and the concentration is maintained within the physiological tolerance range of leukocytes and effectively plays a role.

[0017] Fourth, the implementation of real-time dynamic monitoring and feedback regulation: the image sensor captures dynamic images of the leukocyte stratification interface in the centrifugal container in real time, and transmits high-speed data to the control board. The image processing algorithm built in the control board analyzes the image in real time, identifies the position, thickness and clarity of the stratification interface, and monitors whether there is an abnormal cell aggregation or sedimentation mode. If it is detected that the preset parameters are deviated or stress signs (such as stratification blur, increased cell aggregation) appear, the control board can fine-tune the centrifugal speed, centrifugal time, light parameters (intensity, wavelength), temperature parameters or gas molecule release parameters according to the preset feedback logic, realize the adaptive optimization of the centrifugation process, and ensure that the leukocytes complete the separation in the best physiological state. The temperature sensor synchronously monitors the ambient temperature to provide accurate temperature reference for all the above adjustments.

[0018] Finally, after completing the gradient centrifugation separation, the device automatically stops running. The operator extracts the target leukocyte layer located in the specific density gradient layer through the specific sampling port of the centrifugal container according to the stratification information fed back by the image sensor. The leukocyte layer after the above light biological regulation, precise temperature control and gas signal molecule intervention, its cell function integrity, physiological stability and inflammatory factor release level are significantly better than the leukocytes extracted by the prior art. Subsequently, the extracted leukocytes can be directly used for downstream immunological research, cell therapy or high-quality active ingredient preparation and other applications.

[0019] The beneficial effects of the present application are as follows: The gradient centrifugal leukocyte extraction device and method for inhibiting the release of inflammatory factors provided by the present application significantly reduce the release of inflammatory factors by synergistically inhibiting the stress response of leukocytes during the extraction process from multiple dimensions. First, photobiomodulation enhances the resistance of cells to external pressure from the energy metabolism level by directly affecting mitochondrial function and regulating inflammatory signaling pathways. Second, precise low-temperature control maximally slows down cell metabolic activity and reduces physiological stress caused by mechanical shearing and osmotic pressure changes. Third, the introduction of gas signaling molecules, especially biologically active gases such as nitric oxide, directly intervenes in intracellular signal transduction, inhibiting the key steps of inflammatory factor transcription and release from the molecular level, and has anti-apoptotic and cell-protecting functions. Finally, the real-time monitoring and feedback control system ensures the dynamic optimization of all environmental parameters, so that the entire separation process is always in the most friendly state for leukocytes. The organic combination of these innovative mechanisms enables the present application to obtain high-quality leukocytes with more complete functions, more stable physiological states, and significantly reduced inflammatory factor release, thereby fundamentally improving the application value of leukocyte extraction technology. BRIEF DESCRIPTION OF DRAWINGS

[0020] The present application will be further described below in conjunction with the drawings.

[0021] Figure 1 is a perspective view in the present application; Figure 2 is a schematic view of the internal structure of the fixed cavity in the present application; Figure 3 is a schematic view of the structure of the mounting groove in the present application; Figure 4 is a schematic view of the structure of the second magnetic ring in the present application; Figure 5 is a schematic view of the structure of the cooling assembly and the heating assembly in the present application; Figure 6 is a flowchart of the method of the present application; In the figure: 10, centrifuge body; 11, cover; 12, placement cavity; 13, centrifuge container; 20, mounting groove; 21, illumination lamp; 22, image sensor; 23, temperature sensor; 24, cooling assembly; 25, heating assembly; 26, control panel; 27, adjustment button; 30, gas storage box; 31, fixed cavity; 32, support rod; 33, gas guide pump; 34, gas guide pipe; 35, gas injection hole; 40, support frame; 41, support block; 42, microfiltration membrane; 401, positioning groove; 402, first magnetic ring; 403, second magnetic ring; 404, rubber block. DETAILED DESCRIPTION

[0022] In order to make the technical means, creative features, purposes and effects of the present application easy to understand, the present application will be further described below in conjunction with specific embodiments.

[0023] As Figures 1-5 shown, the gradient centrifugation leukocyte extraction device for inhibiting the release of inflammatory factors described in the embodiments of the present application is constructed on a centrifuge body 10 which is structurally stable and functionally integrated. The centrifuge body 10 is manufactured by high-strength aluminum alloy integral molding process, with an external size of 500 mm in length, 400 mm in width, and 350 mm in height, and a wall thickness of 8 mm, which ensures the structural rigidity and operational stability of the device during high-speed centrifugation operation. The internal space is precisely designed as a cylindrical centrifuge chamber with a diameter of 300 mm and a depth of 200 mm, and the surface is treated by biocompatible passivation to minimize potential contamination of the cell sample. The sidewall of the centrifuge body 10 is rotationally connected to an openable cover 11 through a precision pivot bearing. The pivot bearing uses high-precision self-lubricating ceramic bearings to ensure smooth opening and closing of the cover 11 without wear, and at the same time, it can provide stable mechanical locking force in the closed state. The cover 11 itself is made of reinforced composite material and embedded with sound-absorbing and heat-insulating layers, which can be completely closed during device operation, thereby forming an internal centrifuge chamber that is physically isolated and has highly controlled environmental parameters. This sealing design not only effectively isolates particulate matter, microorganisms and temperature fluctuations in the external environment from interfering with internal operations, but more importantly, it creates the necessary prerequisites for precise control of the internal gas environment, thereby maintaining the constancy of internal environmental parameters and providing an ideal separation microenvironment for leukocytes.

[0024] The inner side of the centrifuge body 10 is precisely and uniformly provided with four independent placement cavities 12 along its circumference. Each placement cavity 12 is a precisely machined cylindrical structure with an inner diameter of 30 mm and a depth of 120 mm, which is optimally designed in size and shape to accurately accommodate and reliably fix a standard 50 ml conical or cylindrical centrifuge container 13, ensuring that the centrifuge container 13 always remains in the pre-set geometric center position during high-speed rotation, thereby avoiding degradation of centrifugal effect or cell damage due to container shaking or positional deviation. The inner wall of the placement cavity 12 is finely polished and sprayed with an anti-static coating to reduce cell adsorption and avoid static charge accumulation.

[0025] Further, the side walls of the placement cavity 12 are symmetrically and equally provided with two groups of mounting slots 20, which are precisely machined by numerical control milling process, with geometric dimensions of 80 mm in length, 15 mm in width, and 10 mm in depth, and the shape is precisely adapted to the external contour of the specific component to be installed, ensuring that the component can be stably embedded and precisely aligned during installation, thereby ensuring the accuracy of the optical path and sensor measurement.

[0026] The inner side wall of the mounting slot 20 is fixedly connected with a lighting lamp 21. The lighting lamp 21 is configured as an array composed of high-brightness and narrow-spectrum light-emitting diode (LED) chips, each group of array containing 20 independently controllable LED units arranged in 2 rows and 10 columns, and the electrical-to-optical conversion efficiency of a single LED is as high as 40%. The LED array can accurately emit monochromatic light of a specific wavelength, and the light output power of each LED unit can be independently adjusted through an independent driving circuit. The specific wavelength can be adjusted in the range of 540 nanometers to 630 nanometers, wherein the 540 nanometer wavelength light is suitable for non-invasive real-time monitoring and imaging of the liquid layering interface and cell sedimentation process in the centrifuge container 13 due to its extremely weak absorption characteristics to hemoglobin, while having minimal impact on the physiological activity of cells and avoiding phototoxicity. The 630 nanometer wavelength light can induce precise regulation of mitochondrial function activity in cells under light irradiation due to its high-efficiency absorption characteristics to Cytochrome c Oxidase (CCO). Specifically, after the 630 nanometer photons are absorbed by CCO, CCO is promoted to change from the oxidized state to the reduced state, thereby enhancing the activity of the electron transport chain, increasing the synthesis efficiency of adenosine triphosphate (ATP), adjusting the production level of reactive oxygen species (ROS) in mitochondria, and inducing the release of endogenous nitric oxide (NO) in cells. Through these complex cascade reactions, effective inhibition of cell stress response and significant reduction of the release of inflammatory factors (such as TNF-α and IL-6) are achieved. The output light power density of the lighting lamp 21 can be precisely controlled by digital signals, with an adjustment range of 10 mW / cm² to 50 mW / cm² and an accuracy of ±0.5 mW / cm², and the light irradiation duration is precisely synchronized with the specific stage in the gradient centrifugation program, ensuring that effective biological regulation is applied during the stage when cells are most susceptible to damage.

[0027] The image sensor 22 is arranged next to the sidewall of the illuminating lamp 21. The image sensor 22 is electrically connected to the driving circuit of the illuminating lamp 21 through a shielded cable to coordinate the timing of illumination and imaging. The image sensor 22 adopts a Sony IMX250 high-resolution global shutter CMOS image sensor module, with an effective pixel array of 2560x1920 pixels and a pixel size of 3.45 microns, having excellent image capture capability and low distortion characteristics. The sensor supports a frame rate of up to 60 frames per second, and can still capture clear dynamic images in a high-speed centrifugal environment. Its spectral response range covers 400 nanometers to 950 nanometers, and can fully capture visible light and part of the near-infrared spectrum. It integrates double sampling technology and advanced noise suppression algorithms, so that it still has excellent low-noise performance (readout noise less than 2e-) in low light conditions, thereby obtaining high signal-to-noise ratio image data. The image sensor 22 is used to capture image information of the liquid layering interface, white blood cell layering process and cell aggregation state in the centrifugal container 13 in real time, and converts it into high-fidelity digital electrical signals through a high-speed MIPICSI-2 interface. The image data is then analyzed in real time by an embedded image processing unit embedded in the control board 26. The unit has image segmentation, edge detection, feature extraction and other algorithms, and can realize non-invasive dynamic evaluation of white blood cell separation efficiency, layering clarity and potential stress indicators (such as cell morphological abnormalities and aggregation degree), providing key visual information for subsequent feedback control.

[0028] A temperature sensor 23 is arranged next to one side of the image sensor 22. The temperature sensor 23 adopts a high-precision A-grade platinum resistance temperature detector (Pt100), with a temperature measurement range of -50°C to 150°C, a measurement accuracy of ±0.1°C in the range of 0°C to 100°C, a response time of less than 1 second, ensuring rapid capture and accurate feedback of temperature changes. The temperature sensor 23 is electrically connected to the control board 26 through a standard RS-485 electrical interface, for real-time monitoring of temperature data of the internal environment of the placement cavity 12 and accurately converting it into digital electrical signals.

[0029] The inner side wall of the placement cavity 12 is symmetrically and stably fixed with a cooling assembly 24 and a heating assembly 25. The cooling assembly 24 adopts a multi-stage thermoelectric cooler array based on the Peltier effect, each array containing 4 Peltier modules of TEC1-12706 model, each module having a single-stage cooling capacity of 80W, a total cooling capacity of 320W, being driven by a direct current voltage (12V / 6A), and realizing precise refrigeration power adjustment through a pulse width modulation (PWM) signal. The heating assembly 25 adopts a PTC ceramic heating element with fast response characteristics, each heating assembly being composed of 2 PTC heaters, each having a maximum heating power of 100W, and a total heating power of 200W, the self-limiting temperature characteristic ensuring safe use. The cooling assembly 24 and the heating assembly 25 are electrically connected to the temperature sensor 23 through independent PID control loops. They work cooperatively with a miniature circulating fan through an integrated high-efficiency liquid cooling heat dissipation structure, the liquid cooling system using a non-toxic ethylene glycol water solution as a cooling medium, cooperating with a miniature direct current circulating pump (flow rate 3L / min) and a miniature plate-fin heat exchanger, to ensure rapid and effective heat transfer and dissipation. According to the temperature signal fed back by the temperature sensor 23 in real time, and in combination with the advanced PID control algorithm preset in the control board 26, the temperature inside the placement cavity 12 is accurately and dynamically adjusted to maintain the temperature inside the placement cavity 12 continuously and stably in the preset physiological suitable range. The suitable range can be set to 4-10℃, and the optimal working temperature can be accurately set to 6℃±0.5℃, aiming to minimize the basal metabolic activity of white blood cells and slow down their physiological response under centrifugal stress, thereby effectively inhibiting the production and release of inflammatory mediators and protecting the physiological integrity of cells.

[0030] The control board 26 is stably fixed inside the sidewall of the centrifuge body 10. The control board 26 adopts a high-performance embedded microcontroller unit (MCU) platform, specifically an STM32H750 series microcontroller based on an ARM Cortex-M7 core, having a main frequency of up to 480MHz, integrating a high-speed digital signal processor (DSP) instruction set and a programmable logic gate array (FPGA) module (such as Xilinx Artix-7), and having a rich set of peripheral interfaces, including multiple high-speed UART, SPI, I2C, CAN and Ethernet interfaces, as well as high-precision 16-bit ADC and DAC modules. The control board 26 is electrically connected to the image sensor 22 and the temperature sensor 23 through a high-speed data bus (for example, MIPI CSI-2 for Camera, SPI for Pt100 ADC) and a dedicated communication protocol, ensuring low delay and high reliability of data transmission.

[0031] Three core functional modules are integrated inside the control board 26: data processing module, control logic module and human-machine interaction module. Data processing module: responsible for receiving and processing high-speed image data stream from image sensor 22 (raw image data is processed by Bayer demosaicing, color correction and noise filtering) and accurate temperature data from temperature sensor 23 (linearization and calibration). This module utilizes the floating-point operation capability of DSP to perform real-time analysis on image data, such as executing OpenCV library-based image segmentation algorithm to accurately identify the layered interface of centrifugal liquid, quantitatively analyze the degree of cell aggregation, and perform sliding average and outlier detection on temperature data to ensure data accuracy and robustness.

[0032] Control logic module: is the "brain" of the entire device, which provides intelligent analysis and decision-making on real-time data provided by the data processing module according to preset gradient centrifugation program, photobiomodulation program, temperature control program and gas signal molecule release program. This module is embedded with multivariable PID control algorithm for precise regulation of the brightness and wavelength of illumination lamp 21, the power of cooling assembly 24 and heating assembly 25, and the gas flow rate and duration of the subsequent gas delivery path. Control commands are output to the corresponding actuators with high precision through DAC (for analog output such as light power) or PWM (for motor drive and heating / cooling power control) signals. In addition, this module also integrates complex sequence control logic and state machine to ensure accurate timing coordination of each link.

[0033] Human-machine interaction module: realized through a 5-inch capacitive TFT LCD touch screen with a resolution of 800x480 pixels, combined with the adjustment button 27 in the form of high-precision rotary encoder exposed on the side wall of the control board 26. The adjustment button 27 is integrated with a press confirmation function, allowing the operator to set and adjust the operating parameters of the control board 26 through an intuitive graphical user interface (GUI) and physical rotary knob, including centrifugal speed (range: 500 RPM to 4000 RPM, step: 10 RPM), centrifugation time (range: 1 minute to 60 minutes, step: 1 minute), target temperature (range: 4℃ to 10℃, step: 0.1℃), light parameters (wavelength 540nm / 630nm selection, intensity 10-50mW / cm², duration 0-30 minutes) and gas molecule release parameters (gas type, concentration 5-200ppm, release time 0-15 minutes), thereby realizing fine and programmable control of the entire white blood cell extraction process environmental parameters.

[0034] The control board 26 is also integrated with a fault diagnosis and safety protection module, which contains multiple sensors (such as vibration sensors, cover locking sensors, overcurrent protection, overtemperature protection, etc.), can monitor the running state of the equipment in real time, and when detecting abnormal conditions deviating from the safety range or preset threshold, trigger an alarm and execute a preset safety shutdown program (for example, emergency stop the centrifugal rotor in an unbalanced state) to ensure the safety of the operator and the equipment.

[0035] The sidewall inside the cover 11 is firmly fixed with a gas storage box 30. The gas storage box 30 is made of medical-grade 316L stainless steel with a wall thickness of 2 mm, and the inside is designed to be airtight under high pressure, with a maximum pressure capacity of 10 MPa. It is filled with 2 liters of high-purity inert carrier gas (such as nitrogen with a purity of 99.999%) mixed with a specific proportion of gas signal molecules. The gas signal molecules can include but are not limited to nitric oxide (NO), hydrogen sulfide (H2S), or carbon monoxide (CO), among which the present application preferably uses nitric oxide (NO) because it has significant anti-inflammatory, anti-apoptotic, and vasodilatory effects on white blood cells at physiological concentrations and is easily metabolized in the body. The concentration of nitric oxide in the premixed gas can be precisely adjusted between 5 ppm and 200 ppm, and calibrated by a high-precision mass flow controller during filling, ensuring that it exerts the maximum biological effect within the physiological tolerance range of white blood cells. The gas storage box 30 is sealed and connected to the input end of the gas guide pump 33 through a standard medical-grade cartridge pneumatic connector, ensuring that the gas transmission path is leak-free.

[0036] The inside of the cover 11 is structured to have a fixed cavity 31. The fixed cavity 31 is made of high-molecular polymer injection molding, and its geometric shape and size are precisely designed to accurately fix the internal sub-components (such as support rods 32, gas guide pumps 33, and gas guide tubes 34), ensuring their structural stability during high-speed rotation of the centrifuge, avoiding component loosening or damage due to vibration. The sidewall of the fixed cavity 31 is fixed with a support rod 32. The support rod 32 is made of aviation-grade 7075 aluminum alloy and has undergone precise structural optimization, adopting a hollow truss structure to achieve lightweight while maintaining high rigidity, with a cross-sectional size of 10x10 mm. The support rod 32 provides reliable mechanical support for the gas guide pump 33, gas guide tube 34, and related sensors (such as micro pressure sensors), and embeds silica gel damping pads to effectively buffer high-frequency vibrations transmitted by the centrifuge rotation or externally, thereby protecting sensitive components.

[0037] The side wall of the support rod 32 is fixed with an air guide pump 33. The air guide pump 33 adopts a NMP05S micro stepping motor driven diaphragm pump produced by the German KNF company, which can realize accurate adjustment of the flow from 0.1 mL / min to 10 mL / min, and the adjustment accuracy is up to ±0.05 mL / min, which ensures the accurate delivery of gas signal molecules. The pump is built-in with a MEMS micro pressure sensor for real-time monitoring of the gas output pressure (range 0-100 kPa, accuracy ±0.5 kPa), and communicates with the control board 26 through a feedback loop, ensuring the stability and accuracy of gas delivery. The input end of the air guide pump 33 is extended to the internal gas outlet end of the gas storage box 30 through a silicone hose (inner diameter 2 mm, wall thickness 1 mm, maximum pressure resistance 0.5 MPa) with excellent pressure resistance and biocompatibility, ensuring the effective extraction of gas signal molecules without leakage.

[0038] The output end of the air guide pump 33 is connected with at least one set of air guide pipe 34 through a micro pneumatic joint. In this embodiment, four independent air guide pipes 34 are used, each corresponding to a placement cavity 12, ensuring uniform distribution of gas in each separation cavity. The air guide pipe 34 is made of polytetrafluoroethylene (PTFE) material with excellent biocompatibility, with an inner diameter of 0.5 mm and an outer diameter of 1.0 mm, which has excellent chemical stability and extremely low active gas adsorption, minimizing gas loss and avoiding pollution. The end of the air guide pipe 34 is uniformly distributed and fixed with a plurality of microporous air injection holes 35. Each air guide pipe end integrates a circular air injection head formed by micro laser drilling technology, which is uniformly distributed with 10 air injection holes, with a single air injection hole diameter of 0.1 mm to 0.3 mm, arranged in an array, ensuring that the gas signal molecules can be uniformly dispersed in the form of extremely fine bubbles into the air environment in the centrifugal chamber inside the centrifuge body 10. This uniform dispersion design aims to ensure that the white blood cells in the centrifuge container 13 can be fully and uniformly exposed to the environment of the gas signal molecules with anti-inflammatory activity during centrifugal separation. The air guide pump 33, the air guide pipe 34 and the air injection hole 35 together constitute a precise gas delivery and dispersion path for accurately releasing the gas signal molecules stored in the gas storage box 30 into the centrifugal environment inside the centrifuge body 10 with a preset flow rate, duration and concentration. These gas molecules quickly fill the centrifugal chamber through gas phase diffusion, and partially dissolve in the centrifugal liquid, thereby achieving the purpose of inhibiting the leukocyte stress response from the molecular level.

[0039] As a preferred embodiment of the present application, the top end of the placement cavity 12 is fixed with a support frame 40 through a precision machining process. The support frame 40 is made of medical-grade 316L stainless steel material, which has good corrosion resistance and mechanical strength after surface passivation treatment, and its size is accurately matched with the placement cavity 12. The top end of the support frame 40 is provided with a detachable support block 41. The support block 41 is precisely injection molded by high-performance polyether ether ketone (PEEK) material, and its external contour is accurately matched with the internal geometry of the support frame 40. Through the innovative combination of quick buckle and self-locking thread connection, it realizes quick installation and disassembly, ensures that it will not loosen during centrifugation, and facilitates the replacement or cleaning of the filter membrane.

[0040] The side wall of the support block 41 is fixed with a microfiltration membrane 42. The microfiltration membrane 42 is made of polyether sulfone (PES) or polyvinylidene fluoride (PVDF) membrane material with a pore size of 0.2 microns to 5 microns. In this embodiment, a PES membrane with a pore size of 2 microns is selected, which has good hydrophilicity, high porosity (>70%) and flow rate (water flux >1000 L / m².h.bar), and can effectively reduce the filtration resistance. The microfiltration membrane 42 further physically filters potential cell debris, incompletely lysed red blood cells and other non-cellular impurities during leukocyte extraction. This secondary filtration mechanism significantly improves the purity of the final leukocyte product, while reducing the impact of these impurities on subsequent immunological analysis or cell therapy applications, ensuring higher quality of the extracted leukocytes. The microfiltration membrane 42 is precisely combined with the internal frame of the support block 41 through hot melt bonding technology, ensuring airtightness and no leakage.

[0041] The side wall of the support frame 40 is provided with an annular positioning groove 401 along its inner periphery. The positioning groove 401 is numerically controlled milling, and its size and shape are used to accurately accommodate and fix specific components, ensuring accurate alignment of the subsequent magnetic ring. The first magnetic ring 402 is fixed inside the side wall of the positioning groove 401. The first magnetic ring 402 is made of high-performance neodymium iron boron permanent magnet material (N52 grade), with an outer diameter of 28 millimeters, an inner diameter of 20 millimeters, a thickness of 3 millimeters, and a surface treated with nickel-copper-nickel three-layer plating to enhance corrosion resistance. Its magnetic field strength can reach 1.2 Tesla on the surface.

[0042] The bottom end of the support block 41 is fixed with a second magnetic ring 403 corresponding to the position of the first magnetic ring 402. The second magnetic ring 403 also adopts high-performance neodymium iron boron permanent magnet material (N52 grade), which is completely the same size as the first magnetic ring 402, and also undergoes nickel-copper-nickel plating treatment. Its polarity is specially designed to be the same as that of the first magnetic ring 402, so that the second magnetic ring 403 and the first magnetic ring 402 form a stable and quickly separable magnetic connection, generating an attractive force of about 15 Newtons in the vertical direction, which is sufficient to firmly fix the centrifugation process, while allowing the operator to exert appropriate force to achieve quick and tool-free disassembly between the support block 41 (together with the microfiltration membrane 42) and the support frame 40, thereby greatly facilitating the replacement or cleaning of the filter membrane and improving the maintainability of the equipment.

[0043] The top end of the support block 41 is fixed with four high-elasticity rubber blocks 404 symmetrically and uniformly. The rubber blocks 404 adopt medical-grade silicone rubber material (Shore A hardness 50 degrees) with excellent shock absorption and buffering performance, and are designed in the shape of a circular truncated cone with a bottom diameter of 15 mm, a top diameter of 10 mm, and a height of 8 mm. They are strategically placed at the position where the support block 41 contacts the top end of the centrifugation container 13, aiming to effectively absorb and attenuate mechanical stress transmitted by centrifugal rotor imbalance, motor vibration or external vibration during the centrifugation process, by converting high-frequency vibration into low-amplitude thermal energy, to minimize the shear stress on white blood cells, thereby significantly reducing cell damage and stress activation, and ensuring cell integrity. In addition, these rubber blocks 404 also have a sealing function, which can form an effective physical seal with the mouth of the container when the centrifugation container 13 is placed and locked, further ensuring the airtightness of the centrifugation chamber, preventing the leakage of gas signaling molecules, and maintaining the stability of the internal environment.

[0044] As shown in Figure 6 The present application also provides a gradient centrifugation white blood cell extraction method for inhibiting the release of inflammatory factors, which is realized by using the above-mentioned device, and the specific steps are designed to work synergistically to achieve the best cell protection effect: Firstly, before the gradient centrifugation leukocyte extraction device is running, the operator sets the expected centrifugation parameters (including rotation speed, time, etc.) and various environmental adjustment parameters (such as temperature, light wavelength, intensity, duration, and gas molecule type, concentration, release time, etc.) through the human-computer interaction interface on the control panel 26. Subsequently, the internal environmental temperature of the placement cavity 12 of the device is pre-adjusted. The control panel 26 instructs the cooling assembly 24 and the heating assembly 25 to work cooperatively according to the preset program, and the power output of each is controlled by the precise PID algorithm. This process rapidly and accurately controls and maintains the temperature in the placement cavity 12 in the physiological low-temperature range of 4°C to 10°C, which is preset to 6°C±0.5°C in this embodiment. This low-temperature environment can significantly reduce the basal metabolic rate of cells and slow down the speed of enzymatic reactions, thereby providing a low-stress initial environment for the subsequent centrifugation separation process and laying a foundation for the physiological stability of leukocytes. Generally, the temperature stabilization process takes about 5 to 10 minutes. Secondly, the blood sample to be separated (for example, 20 milliliters of freshly collected human whole blood) and the pre-prepared gradient centrifugation liquid (for example, Ficoll-Paque PLUS lymphocyte separation medium, density 1.077 g / mL, 20 milliliters) are carefully injected into the pre-cooled 50 milliliter conical centrifuge container 13 in a specific ratio. When injecting, the gradient centrifugation liquid is slowly injected along the tube wall into the bottom of the centrifuge tube, and then the blood sample is carefully placed above the gradient liquid to avoid violent mixing of the two, so as to ensure the formation of a clear and stable density gradient layer in the centrifuge container 13. Then, the filled centrifuge container 13 is stably placed in the preset position inside the device placement cavity 12, ensuring its vertical alignment. The operator closes the cover 11 and locks it. The device is then started and performs the pre-programmed gradient centrifugation program. The program usually includes multiple stages, such as: the first stage (low-speed centrifugation, such as 800 RPM, 5 minutes, for preliminary cell sedimentation), the second stage (medium-speed centrifugation, such as 1800 RPM, 20 minutes, for density gradient separation), and the third stage (low-speed braking, such as 500 RPM, 2 minutes, for smooth stopping). Each stage is set with a specific centrifugation speed, centrifugation time, and time point linked with the temperature control system.

[0045] After the centrifugation program is started, the method performs the following multi-dimensional synchronous adjustment strategy at a specific stage of centrifugal separation to achieve fine regulation of the physiological state of leukocytes: First, photobiomodulation is implemented: the control board 26 precisely controls the array of illumination lamps 21 according to the progress of the centrifugation procedure and the real-time feedback of cell status from the image sensor 22. When the centrifugation enters the critical stage of cell sedimentation and stratification formation (usually within 5-25 minutes after the start of centrifugation), the control board 26 instructs the illumination lamps 21 to emit monochromatic light of a specific wavelength, which can be 630 nanometers or 540 nanometers. In this embodiment, to inhibit the release of inflammatory factors, it is preferred to continuously irradiate the leukocyte suspension in the centrifuge container 13 with a set light power density (for example, 20 mW / cm², ±1 mW / cm²) when the centrifugation enters the medium-speed stage (1800 RPM). After the 630 nanometer wavelength light is efficiently absorbed by cytochrome c oxidase (CCO) in the mitochondria of leukocytes, it triggers a change in the CCO conformation, which in turn enhances the activity of the mitochondrial electron transport chain and promotes the synthesis of adenosine triphosphate (ATP). The increase in ATP levels helps cells maintain normal physiological functions, enhances the stability of the cell membrane, and provides energy to resist stress caused by centrifugal shear force. At the same time, light can finely regulate the level of reactive oxygen species (ROS) production, avoiding the excessive accumulation of ROS that can cause oxidative stress damage. More importantly, this wavelength of light, through a series of complex molecular mechanisms, can inhibit the activation of key inflammatory signaling pathways such as NF-κB, thereby significantly reducing the early release of inflammatory factors such as tumor necrosis factor-alpha, TNF-alpha; interleukin-6, IL-6. The duration of light exposure can be dynamically adjusted according to the stage of centrifugation, and the typical duration is 10-30 minutes, for example, continuously irradiating for 20 minutes during the entire medium-speed centrifugation stage to ensure that the photobiomodulation effect is fully achieved. The uniformity of light exposure is ensured by the internal light path design and LED array arrangement, and the irradiation area covers the entire area where leukocytes may be distributed.

[0046] Second, maintain precise temperature control: the temperature sensor 23 continuously monitors the temperature inside the placement cavity 12 in real time at a frequency of 1 second / second. After receiving the temperature data, the control board 26 accurately adjusts the cooling or heating power of the cooling assembly 24 and the heating assembly 25 through the built-in precisely optimized PID control algorithm. For example, when the temperature is higher than the set value (such as 6.5°C), increase the PWM duty cycle of the cooling assembly 24; when the temperature is lower than the set value (such as 5.5°C), increase the PWM duty cycle of the heating assembly 25. This two-way, closed-loop temperature control ensures that the white blood cells inside the centrifugal container 13 are always at the pre-set, strictly constant physiological suitable temperature (for example, 6°C±0.2°C). This constant and precise low temperature environment can effectively inhibit the excessive metabolism and non-specific stress response of cells under centrifugal shear force, further reduce the synthesis and release of inflammatory mediators, and at the same time maximize the maintenance of cell membrane integrity and functional activity. Temperature fluctuations are strictly controlled within a very small range, providing an extremely stable physical environment for cells.

[0047] Third, introducing gaseous signaling molecule intervention: the control board 26 instructs the gas guide pump 33 to start at specific critical stages of the gradient centrifugation process, such as at the early stage of cell sedimentation (about 5 minutes after the start of centrifugation) or at the stage of layer formation (about 15-25 minutes after the start of centrifugation). The gas guide pump 33 extracts and delivers a high-purity mixture of nitric oxide gas signaling molecules from the gas storage box 30 at a preset precise flow rate (e.g., 5 mL / min, with an adjustment accuracy of ±0.05 mL / min). The gas is uniformly dispersed in the form of micron-sized bubbles into the air environment of the centrifugation chamber through the gas guide tube 34 and the array of gas injection holes 35, and diffuses in the gas phase and dissolves in the centrifugation liquid, so that the cells in the centrifugation container 13 can be fully exposed to a nitric oxide environment with a set concentration (e.g., 50 ppm). The nitric oxide activates the soluble guanylate cyclase (sGC) in the cells, catalyzes the conversion of guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP), thereby increasing the intracellular cGMP level. As an important second messenger, cGMP activates downstream signaling pathways such as protein kinase G (PKG), thereby exerting its multiple biological effects: including anti-inflammatory effects (inhibiting the activity of transcription factors such as NF-κB and AP-1, reducing the expression of inflammatory factor genes), anti-apoptotic effects (stabilizing mitochondrial membrane potential, inhibiting caspase activation), antioxidant effects (directly scavenging ROS or upregulating antioxidant enzyme activity), and inhibiting leukocyte adhesion and activation, thereby inhibiting the release of inflammatory factors induced by leukocytes under centrifugal stress at the molecular level. The duration of release of gaseous signaling molecules is usually 5-15 minutes, and in this embodiment it is set to 10 minutes, ensuring that the gas concentration is within the physiological tolerance of leukocytes and effectively exerts its effects. The uniform dispersion of the gas is optimized by the carefully designed gas injection hole array and the geometry of the centrifugation chamber, so that all cells in the centrifugation tubes can receive the same intensity of gas intervention.

[0048] Fourth, real-time dynamic monitoring and feedback regulation: The image sensor 22 continuously captures dynamic images of the leukocyte layering interface inside the centrifuge container 13 at a frequency of 10 frames per second during the centrifugation process and transmits the images to the control board 26 at high speed. The image processing algorithm built into the control board 26 analyzes the images in real time, accurately identifies the position, thickness (such as the thickness of the lymphocyte layer), and clarity of the layering interface, and monitors whether there are abnormal cell aggregation or sedimentation patterns (for example, red blood cells do not fully sediment, white blood cell layer diffusion, or cell agglutination). For example, the layering clarity is evaluated by calculating the interface edge gradient and average pixel gray value, and the image recognition algorithm is used to quantify the cell morphology and aggregation index. If it is detected that the deviation from the preset parameters or the stress signs (for example, the layering blurring degree increases by more than 10%, the cell aggregation increases by more than 20%), the control board 26 can adjust the centrifugation speed (fine-tune ±50 RPM), the centrifugation time (extend or shorten 2-5 minutes), the light parameter (intensity increase 5 mW / cm² or wavelength switching), the temperature parameter (target temperature fine-tune ±0.5℃) or the gas molecule release parameter (gas concentration increase 10 ppm or duration extension 3 minutes) according to the preset feedback logic (for example, based on fuzzy control or decision tree of expert system). This adaptive feedback regulation mechanism ensures that the entire centrifugation process is always in the best physiological state for white blood cells, thereby maximizing the separation efficiency and physiological integrity of white blood cells. The temperature sensor 23 synchronously monitors the ambient temperature, providing accurate temperature reference and compensation for all the above adjustments.

[0049] Finally, after completing the gradient centrifugation separation, the device automatically stops running and brakes at a very low deceleration rate to avoid shear force disturbance to the layered cells. According to the final layering information fed back by the image sensor 22 at the end of the separation (displayed on the display screen, indicating the exact position and thickness of the target white blood cell layer), the operator uses a specific sampling port (for example, using a long-handled pipette or a precision syringe) of the centrifuge container 13 to accurately extract the target white blood cell layer (for example, the lymphocyte and monocyte layer) located in a specific density gradient layer. The white blood cell layer, through the synergistic effect of the above light biological regulation, precise temperature control, and gas signal molecule intervention, has significantly better cell function integrity, physiological stability, and inflammatory factor release level than the white blood cells extracted by the prior art. Subsequently, the extracted white blood cells have higher activity and purity, and can be directly used for downstream immunological research, preparation of cell therapy products, high-quality active ingredient extraction, or in vitro diagnosis of high-demand applications.

[0050] The device and method provided by the present application fundamentally inhibit the stress response of white blood cells during extraction through the synergistic effect of multiple dimensions, thereby significantly reducing the release of inflammatory factors. First, photobiomodulation enhances the resistance of cells to external stress from the energy metabolism level by directly affecting mitochondrial function and directly regulating key inflammatory signaling pathways such as NF-κB. Second, precise low-temperature control maximizes cell metabolic activity, reduces physiological stress caused by mechanical shear and osmotic pressure changes, and maintains the integrity of cell membranes and cytoskeleton. Third, the introduction of gas signaling molecules such as nitric oxide directly intervenes in intracellular signal transduction, inhibiting key steps in the transcription and release of inflammatory factors from the molecular level, and has significant anti-apoptotic, antioxidant, and cell protective functions. Finally, the real-time monitoring and feedback control system ensures dynamic optimization of all environmental parameters, ensuring that the entire separation process is always in the most favorable state for white blood cells. The organic combination of these innovative mechanisms enables the present application to obtain high-quality white blood cells with more complete functions, more stable physiological conditions, and significantly reduced inflammatory factor release, thereby fundamentally improving the application value and conversion potential of white blood cell extraction technology.

[0051] Examples This example aims to verify the improvement effect of the "Gradient centrifugal white blood cell extraction device and method for inhibiting the release of inflammatory factors" disclosed in the present application on the functional integrity, physiological stability, and inflammatory factor release level of white blood cells in actual application.

[0052] Experimental materials and equipment Device of the present application: a gradient centrifugal white blood cell extraction device for inhibiting the release of inflammatory factors according to the detailed description in the above "Detailed description". Setting parameters: Chamber temperature: 6.0°C ± 0.2°C. Illumination parameters: 630 nm wavelength, light power density 25 mW / cm², continuous irradiation for 20 minutes from the 5th minute after the start of centrifugation. Gas signaling molecules: nitric oxide (NO), concentration preset to 80 ppm, continuous release for 12 minutes from the 8th minute after the start of centrifugation, flow rate 5 mL / min. Centrifugation program: Pre-centrifugation: 800 RPM, 5 minutes. Separation centrifugation: 1800 RPM, 20 minutes. Braking: 500 RPM, 2 minutes. Real-time feedback: image sensors adjust the centrifugation speed (± 50 RPM) and light intensity (± 5 mW / cm²) in real time according to the layering clarity and cell aggregation degree. Blood samples: 40 mL of venous whole blood from 5 healthy adult volunteers (25-35 years old, no underlying diseases) were collected in EDTA anticoagulant tubes. The samples were processed within 2 hours after collection.

[0053] Gradient centrifugation solution: Ficoll-Paque PLUS Lymphocyte Separation Medium (density 1.077 g / mL).

[0054] Centrifugation container: 50 mL standard conical sterile centrifuge tube. Analytical reagents and equipment: Leukocyte counter: Sysmex KX-21N. Cell viability detection kit: Trypan blue staining method. Cell activity assay: CellTiter-Glo® Luminescent Cell Viability Assay (ATP detection). Inflammatory factor detection: Human TNF-a and IL-6 Quantikine ELISA Kit (R&D Systems).

[0055] Flow cytometry: BD FACSCalibur™ (for analyzing CD45+ / CD14- cell purity).

[0056] RNA extraction kit and RT-qPCR system (for detecting inflammation-related gene expression, such as NFKBIA). Experimental steps Sample preparation: Each blood sample was divided into two parts, each 20 mL, for the device and the comparative experiment. Carefully stack 20 mL of blood sample on top of 20 mL of Ficoll-Paque PLUS separation solution, a total of 40 mL mixture per tube. Device operation: Place the prepared centrifuge tube in the pre-cooled placement cavity of the device. Start the device and perform the above-mentioned centrifugation program and synchronous regulation strategy (light biological regulation, precise temperature control, gas signal molecule intervention, real-time monitoring and feedback regulation). After centrifugation, use a precision pipette to carefully aspirate the mononuclear cell layer and lymphocyte layer at the interface in a biological safety cabinet. Transfer the extracted cells to a new centrifuge tube and wash twice with physiological saline (400g, 5 minutes) to remove residual separation solution. Resuspend the cells in RPMI 1640 medium for subsequent analysis. Data collection and analysis: Leukocyte purity: Take a portion of the cell suspension and perform CD45+ / CD14- cell staining using a flow cytometer to calculate the percentage of leukocyte purity. Cell viability: Take a portion of the cell suspension and perform trypan blue staining to count the percentage of viable cells.

[0057] Cell activity (ATP level): Take a portion of the cell suspension and use the CellTiter-Glo® Luminescent Cell Viability Assay to detect ATP content, reflecting cell metabolic activity.

[0058] Inflammatory factor release: Take a portion of the cell suspension and incubate it in serum-free RPMI 1640 medium for 2 hours, collect the supernatant, and use an ELISA kit to detect the concentrations of TNF-α and IL-6. Gene expression: Extract total RNA from the cells, perform reverse transcription, and then detect the relative expression level of the NFKBIA (NF-κB inhibitor α) gene by RT-qPCR. Comparative example To objectively evaluate the technical effects of the present application, a control group was set up using the existing conventional gradient centrifugation leukocyte extraction method for comparison. Control equipment and method: Conventional centrifuge: a commercially available conventional benchtop high-speed centrifuge without temperature control, without light intervention, and without gas intervention. Setting parameters: Temperature: room temperature (22℃ ± 2℃) without active temperature control. Light: no light intervention. Gas: no gas signal molecule intervention. Centrifugation program: Pre-centrifugation: 800 RPM for 5 minutes. Separation centrifugation: 1800 RPM for 20 minutes. Braking: 500 RPM for 2 minutes. No real-time feedback control. Blood samples, gradient centrifugation liquid, centrifuge containers, analysis reagents and equipment: exactly the same as in this example, ensuring the comparability of experimental conditions. Experimental steps: Sample preparation: the same as in this example. Conventional centrifuge operation: The prepared centrifuge tube was placed in the centrifugal cavity of a conventional centrifuge. The conventional centrifuge was started and the same centrifugation program was performed without any additional regulation of environmental parameters.

[0059] After centrifugation, the white blood cell layer was aspirated in the same manner and washed twice. The cells were resuspended in RPMI 1640 medium for subsequent analysis. Data collection and analysis: The same methods were used to detect white blood cell purity, cell viability, cell activity, inflammatory factor release, and gene expression as in this example.

[0060] Comparison of experimental results and data The experimental data of 5 volunteer samples were summarized and averaged, and the results are shown in the following table:

[0061] From the above experimental results, it can be seen that the gradient centrifugation white blood cell extraction device and method for inhibiting inflammatory factor release provided by the present application has significant advantages in multiple key indicators: White blood cell purity: The purity of white blood cells extracted by the device of the present application reached 96.8%±0.7%, which was about 3.5% higher than that of 93.5%±1.2% extracted by the conventional centrifuge. This indicates that the precise temperature control, optimized centrifugation program and potential microfiltration membrane auxiliary function of the present application can more effectively separate the target cell population and reduce the mixing of red blood cells, platelets or other impurities. Cell viability: The viability of white blood cells extracted by the present application was as high as 98.2%±0.5%, while the conventional method was only 92.1%±1.5%. This means that the physical damage and physiological stress to cells during the entire extraction process have been greatly alleviated, the integrity and function of the cell membrane are better maintained, and the cell death rate is significantly reduced. Cell activity (ATP level): The average relative ATP level of white blood cells extracted by the present application was 1.85, which was almost 1.85 times that of the conventional method (1.00). ATP is the energy currency of cells, and its content directly reflects the metabolic activity and health status of cells. This result strongly proves that photobiomodulation (630 nm light activation of CCO) and precise low temperature control can significantly improve the energy reserves and metabolic function of white blood cells, making them maintain higher physiological activity after extraction, providing more functional cells for subsequent applications. Inflammatory factor release: This is one of the core advantages of the present application. The amount of TNF-a released by the leukocytes extracted by the present application is only 35.6 ± 4.1 pg / mL after 2 hours of culture, while the amount of TNF-a released by the conventional method is as high as 98.2 ± 9.5 pg / mL, which is reduced by about 63.8%. Similarly, the amount of IL-6 released is significantly reduced from 78.5 ± 7.8 pg / mL to 28.1 ± 3.5 pg / mL, with a reduction of about 64.2%. This clearly shows that the synergistic effect of photobiomodulation, precise temperature control, and gas signal molecule (NO) intervention can effectively inhibit the inflammatory response of leukocytes under centrifugal stress and reduce the production and secretion of pro-inflammatory cytokines (such as TNF-a and IL-6). Relative expression of NFKBIA gene: The NFKBIA gene encodes the NF-κB inhibitor IκBα, and an increase in its expression is usually associated with the inhibition of the NF-κB pathway. The relative expression of the NFKBIA gene of the cells extracted by the present application is 1.52, which is higher than 0.88 of the conventional method, with an increase of 72.7%. This confirms the effectiveness of the present application in inhibiting the inflammatory signaling pathway at the gene expression level, i.e., by up-regulating the expression of IκBα, preventing NF-κB from entering the nucleus and activating the transcription of inflammation-related genes, thereby reducing the overall level of inflammatory factors. In summary of the above experimental data, the gradient centrifugation leukocyte extraction device and method proposed by the present application successfully solves the long-standing problem of strong physiological stress response and high inflammatory factor release of leukocytes in the extraction process in the prior art through the organic integration and synergistic effect of multiple mechanisms such as photobiomodulation, precise temperature control, gas signal molecule intervention, and real-time monitoring and feedback regulation. The obtained leukocytes not only perform excellently in purity and viability, but more importantly, their physiological state is more stable, with significantly reduced stress response and inflammatory factor release. This makes the extracted leukocytes have higher reliability, safety, and clinical translation value in high-demand biomedical applications such as immunological research, cell therapy, and drug screening, realizing a revolutionary innovation of leukocyte extraction technology.

[0062] The basic principles, main features, and advantages of the present application are shown and described above. Those skilled in the art should understand that the present application is not limited by the above examples, and the above examples and descriptions in the specification are only to illustrate the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the claimed present application. The scope of protection of the present application is defined by the appended claims and their equivalents.

Claims

1. A gradient centrifugation leukocyte extraction device for inhibiting the release of inflammatory factors, comprising a centrifuge body (10); characterized in that: It also includes: a cover (11), which is rotatably connected to the side wall of the centrifuge body (10) via a pivot bearing, and the cover (11) can be closed during equipment operation to ensure the formation of a physically isolated and environmentally stable internal centrifuge chamber; and a placement cavity (12), at least one set of the placement cavities (12) are uniformly arranged along the circumference of the inner side of the centrifuge body (10), and the internal structure of the placement cavity (12) is designed to accurately accommodate and reliably fix conical or cylindrical centrifuge containers (13). The lighting lamp (21) has a mounting groove (20) on the side wall of the placement cavity (12), and the lighting lamp (21) is fixed to the inner side wall of the mounting groove (20). The lighting lamp (21) is configured as a light-emitting diode array with multi-spectral emission capability, which can emit monochromatic light of a specific wavelength. The specific wavelength can be adjusted in a range of 540 nm to 630 nm. The output light power density of the lighting lamp (21) can be dynamically adjusted according to the preset centrifugation stage and cell state. Image sensor (22), the image sensor (22) is provided on the side wall adjacent to the lighting lamp (21), the image sensor (22) and the lighting lamp (21) are electrically connected through an electrical signal cable, and are used to capture image information of the liquid layer interface, cell layer sedimentation process and cell aggregation state inside the centrifuge container (13) in real time; Temperature sensor (23): The temperature sensor (23) is provided on one side of the image sensor (22) for real-time monitoring of the temperature data of the internal environment of the placement cavity (12); The cooling component (24) and the heating component (25) are symmetrically and securely fixed to the inner wall of the placement cavity (12). The cooling component (24) and the heating component (25) are electrically connected to the temperature sensor (23) through an independent control circuit. The gas storage box (30) is securely fixed to the side wall inside the cover (11), and the gas storage box (30) is filled with an inert carrier gas and a mixture of gas signal molecules in a specific ratio. An air pump (33) is provided. A support rod (32) is fixed to the side wall of the fixed cavity (31) inside the cover (11). The air pump (33) is fixed to the side wall of the support rod (32). The input end of the air pump (33) extends to the internal gas outlet end of the gas storage box (30) through a pressure-resistant hose. The output end of the air pump (33) is connected to at least one set of air pipes (34) through a micro pneumatic connector. The end of the air pipe (34) is evenly distributed and fixed with multiple micro-hole jet holes (35). The air pump (33), the air pipe (34) and the jet holes (35) together form a precise gas delivery and diffusion path, which is used to accurately release the gas signal molecules stored in the gas storage box (30) into the centrifugal environment inside the centrifuge body (10) at a preset flow rate, duration and concentration. The control is performed by the control board (26) to achieve effective inhibition of leukocyte stress response.

2. The gradient centrifugation leukocyte extraction device for inhibiting the release of inflammatory factors according to claim 1, characterized in that, The control board (26) is securely fixed inside the side wall of the centrifuge body (10). The control board (26) has an adjustment button (27) rotatably mounted on its side wall. The control board (26) is electrically connected to the image sensor (22) and the temperature sensor (23) via a high-speed data bus and a dedicated communication protocol. The control board (26) integrates a data processing module, a control logic module, and a human-machine interaction module. It is used to receive and process high-speed image data streams from the image sensor (22) and precise temperature data from the temperature sensor (23), and to precisely control the brightness and wavelength of the lighting lamp (21) and the power of the cooling component (24) and the heating component (25) according to a preset program.

3. The gradient centrifugation leukocyte extraction device for inhibiting the release of inflammatory factors according to claim 1, characterized in that, The lighting lamp (21) is configured as an array of high-brightness, narrow-spectrum light-emitting diode chips.

4. The gradient centrifugation leukocyte extraction device for inhibiting the release of inflammatory factors according to claim 1, characterized in that, The image sensor (22) is a global shutter CMOS module with a frame rate of 60 frames / second and a spectral response range of 400-950 nanometers. It transmits data through the MIPICSI-2 interface. The control board (26) has an embedded image processing unit.

5. The gradient centrifugation leukocyte extraction device for inhibiting the release of inflammatory factors according to claim 1, characterized in that, The temperature sensor (23) is a Pt100 platinum resistance thermometer with a temperature range of -50 to 150°C and an accuracy of ±0.1°C within 0 to 100°C; the cooling component (24) is an array containing four TEC1-12706 Peltier modules; the heating component (25) consists of two PTC heaters, each with a power of 100W; it can maintain the temperature of the placement chamber (12) at 4-10°C, with an optimal temperature of 6°C ±0.5°C.

6. The gradient centrifugation leukocyte extraction device for inhibiting the release of inflammatory factors according to claim 1, wherein the control board (26) adopts an STM32H750 series microcontroller platform based on the ARM Cortex-M7 core, with a main frequency of up to 480MHz, and integrates a high-speed digital signal processor instruction set and a programmable gate array module. The side wall of the control board (26) is provided with an adjustment button (27) in the form of a high-precision rotary encoder (27). The adjustment button (27) has a press confirmation function and is used in conjunction with a 5-inch capacitive TFTLCD touch screen with a resolution of 800x480 pixels, for the operator to set and adjust the operating parameters of the control board (26).

7. The gradient centrifugation leukocyte extraction device for inhibiting the release of inflammatory factors according to claim 1, characterized in that, The gas storage box (30) is made of medical grade 316L stainless steel with a wall thickness of 2 mm and a maximum pressure of 10 MPa. It contains 2 liters of carrier gas and a mixture of gas signal molecules. The gas pump (33) has a flow rate adjustment range of 0.1-10 mL / min and an accuracy of ±0.05 mL / min. The gas delivery tube (34) is made of PTFE with an inner diameter of 0.5 mm and an outer diameter of 1.0 mm. The jet nozzle (35) has a diameter of 0.1-0.3 mm.

8. The gradient centrifugation leukocyte extraction device for inhibiting the release of inflammatory factors according to claim 1, characterized in that, A support frame (40) is fixed to the top of the placement cavity (12); a support block (41) is provided at the top of the support frame (40); a microfiltration membrane (42) is fixed to the side wall of the support block (41); a positioning groove (401) is provided on the side wall of the support frame (40); a first magnetic ring (402) is fixed to the side wall of the positioning groove (401); a second magnetic ring (403) is fixed to the bottom of the support block (41); the second magnetic ring (403) and the first magnetic ring (402) are connected by magnetic connection; and rubber blocks (404) are symmetrically fixed to the top of the support block (41).

9. A gradient centrifugation method for extracting leukocytes by inhibiting the release of inflammatory factors, applicable to the gradient centrifugation leukocyte extraction device for inhibiting the release of inflammatory factors as described in any one of claims 1-8, characterized in that, Includes the following steps: S1. Equipment pre-adjustment: The control panel (26) instructs the cooling component (24) and the heating component (25) to work together to precisely control and maintain the temperature in the placement chamber (12) within the physiological low temperature range of 4°C to 10°C. S2. Sample preparation and placement: The blood sample to be separated and the gradient centrifugation solution are injected into the centrifugation container (13) in a specific ratio. Then, the centrifugation container (13) is placed securely in the preset position inside the placement chamber (12). The device is started and the pre-programmed gradient centrifugation program is executed. S3. Synchronous regulation of the centrifugation process: Implementation of synchronous regulation strategies at specific stages of centrifugation separation, including photobiological regulation, maintaining precise temperature control, introducing gaseous signaling molecules for intervention, and implementing real-time dynamic monitoring and feedback regulation. S4. After the gradient centrifugation is completed, the equipment will automatically stop running. The operator will accurately extract the target white blood cell layer located in the specific density gradient layer based on the layer information fed back by the image sensor (22).

10. The gradient centrifugation leukocyte extraction method for inhibiting the release of inflammatory factors according to claim 9, characterized in that, The photobiological regulation implemented in S3 is as follows: the control board (26) controls the array of lighting lamps (21) to emit monochromatic light of a specific wavelength (630 nm or 540 nm) according to the progress of the centrifugation program and the feedback of cell status, and continuously irradiates the white blood cell suspension in the centrifugation container (13) with a light power density of 10 mW / cm² to 50 mW / cm² for 10 to 30 minutes. Maintaining precise temperature control specifically involves: temperature sensor (23) continuously monitoring the temperature inside placement cavity (12) in real time; control board (26) adjusting the power of cooling component (24) and heating component (25) through PID control algorithm to maintain the temperature at 6℃±0.5℃. The specific intervention of gas signal molecules is as follows: the control board (26) commands the gas pump (33) to start at a specific stage of centrifugation, and draws and delivers a mixture of gas signal molecules from the gas storage box (30) at a preset flow rate. The gas signal molecules are evenly diffused into the centrifuge chamber through the array of gas pipe (34) and jet holes (35), and the release duration is 5 to 15 minutes. The implementation of real-time dynamic monitoring and feedback control is as follows: the image sensor (22) captures dynamic images of the leukocyte layering interface in the centrifuge container (13) in real time and transmits them to the control board (26). The control board (26) analyzes the images in real time. If an abnormality is detected, the centrifugation speed, time, light parameters, temperature parameters or gas molecule release parameters are finely adjusted.

Images