An integrated method for screening enrichment and activity modification of stem cell-rich micro-lipid fraction

Abstract

The application relates to the field of biological tissue engineering and discloses an integrated method for screening and enriching stem cell-rich micro-fat components and modifying activity, which comprises the following synchronous and cooperative procedures: fat tissue micronization and crushing, fat impurity gradient screening and enrichment, and in-situ mild modification of activity; the method is continuously completed in a closed and sterile preparation device, there is no material transfer and no ex vivo standing transition between procedures, and a biological activity protection system required for modification is in-situ supplied through a built-in micro-release module of the device. The built-in micro-release module can accurately proportion and release a moisturizing and anti-apoptosis composite active factor, in-situ mild modification of the micro-fat particles can be realized, the active factor can be uniformly penetrated into the gaps of the fat particles, and the survival rate of the fat stem cells and the complete retention rate of the extracellular matrix are significantly improved; compared with traditional micro-fat which only has a single performance of filling and shaping, the micro-fat component prepared by the method has excellent soft tissue filling effect and strong cell regeneration.

Description

Technical Field

[0001] This invention relates to the field of bio-tissue engineering technology, specifically to an integrated method for screening, enriching, and modifying the activity of micro-lipid components rich in stem cells. Background Technology

[0002] Autologous fat grafting is one of the core technologies in the fields of bioengineering, medical aesthetics, and clinical wound repair. Compared with artificial fillers and allogeneic implants, autologous fat has outstanding advantages such as excellent biocompatibility, no immune rejection, convenient harvesting, and natural tissue feel. Furthermore, high-quality fat components are rich in adipose-derived stem cells, extracellular matrix, and various active regenerative factors. These components not only achieve the shaping effect of filling soft tissue depressions but also participate in skin collagen regeneration, barrier repair, and wound healing. It is widely used in clinical scenarios such as facial fine line repair, improvement of photo-aged skin, and repair of soft tissue defects, possessing extremely high clinical application value and market potential.

[0003] Existing traditional preparation processes have many inherent technical defects in practical applications, severely restricting the quality of micro-fat products and their clinical therapeutic effects. Firstly, traditional static filtration and centrifugation purification methods can only roughly remove large connective tissue impurities, failing to accurately screen and enrich high-activity, high-stem-density high-quality micro-fat components. They are also ineffective at removing trace amounts of inert impurities such as free inert oils, broken blood cells, and fine fibrous debris. The resulting micro-fat has low purity and poor component uniformity, and residual inert impurities can easily lead to post-transplant inflammatory reactions and uneven absorption. Secondly, the mechanical crushing and purification processes in traditional methods often employ high-shear, high-intensity mechanical treatments, and the entire process cannot maintain a mild physiological environment suitable for stem cell survival. This easily causes damage to the capsule of adipose-derived stem cells and loss of activity, resulting in high apoptosis rates and low survival rates, significantly weakening the regenerative and repair properties of the micro-fat.

[0004] In summary, current stepwise microfat preparation processes cannot simultaneously achieve component purity, particle uniformity, stem cell activity, and regenerative repair performance. The prepared microfat can only achieve basic shaping and filling functions, lacking the core activity for skin repair, collagen regeneration, and wound repair. This results in a series of technical bottlenecks, including cumbersome procedures, high risk of contamination, low cell survival rate, limited product performance, and unstable clinical effects. Currently, the industry lacks a microfat preparation technology that can simultaneously achieve micronization, screening and enrichment, and activity modification, with in-situ gentle modification and a completely sterile, closed-loop process. This makes it difficult to meet the clinical application needs of high-precision, high-activity, and multifunctional autologous microfat. Therefore, there is an urgent need to develop a novel integrated method for screening, enriching, and modifying stem cell-rich microfat components to address the many shortcomings of existing technologies. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides an integrated method for screening, enriching, and modifying the activity of micro-lipid components rich in stem cells, thus solving the problems mentioned in the background section.

[0006] To achieve the above objectives, the present invention provides the following technical solution: an integrated method for screening, enriching, and modifying micro-lipid components rich in stem cells, comprising the following simultaneous and coordinated steps: micronized adipose tissue disruption, gradient screening and enrichment of adipose impurities, and in-situ mild modification; the entire method is completed continuously within a closed, sterile preparation device, with no material transfer or in vitro static transition between steps, and the bioactive protection system required for modification is supplied in-situ through a built-in micro-release module, eliminating the need for additional exogenous active reagents after preparation; During the in-situ active and mild modification process, the micro-release module precisely releases moisturizing and anti-apoptotic active factors adapted to the micro-fat particles. The two types of active factors penetrate evenly into the interstitial space of the micro-fat particles, simultaneously completing the retention and enrichment of high-quality micro-fat components rich in adipose stem cells and extracellular matrix, while removing inert oil impurities, connective tissue fragments, and inert particles of blood cell sediment that have no regenerative activity.

[0007] Preferably, the closed aseptic preparation device integrates at least a microparticle fragmentation chamber, a gradient screening and retention chamber, and a micro-release modification chamber, forming a three-chamber interconnected structure. The air pressure, temperature, and fluid flow rate inside the three chambers are linked and regulated to maintain a mild physiological environment of 32-36°C throughout the process, avoiding stem cell damage and apoptosis caused by high temperature and high-speed shearing.

[0008] Preferably, the adipose tissue microparticle fragmentation process adopts a low-shear reciprocating shear fragmentation structure, with the shear gap controlled at 100-300 μm, a single fragmentation cycle duration of 5-12 s, and the fragmented output micro-fat particles with a concentrated distribution of 150-280 μm in size, ensuring that the adipose stem cells and extracellular matrix inside the particles are completely preserved and that the cell membrane does not rupture.

[0009] Preferably, the gradient screening and enrichment process adopts a two-stage gradient sieving structure; the first-stage sieving has a pore size of 400 μm, which traps large connective tissue, fatty fiber, and inert impurities and discharges them in a directional manner; the second-stage sieving has a pore size of 120 μm, which traps and enriches micro-fatty components with a particle size of 120-300 μm and high stem cell density, while free oil with a particle size of less than 120 μm and broken blood cell waste liquid are discharged simultaneously under negative pressure, so as to achieve real-time separation of high-quality active components and inert impurities.

[0010] Preferably, the device has a built-in micro-release module that is a porous microsphere-filled sustained-release unit, with the microspheres pre-loaded with a composite bioactive protection system. The composite bioactive protection system is composed of moisturizing active components and anti-apoptotic active components, and the distribution ratio of each component in the system is dynamically matched to the release rate based on the cell density of the micro-lipid particles obtained by secondary sieving.

[0011] Preferably, the moisturizing active factor comprises hyaluronic acid oligosaccharide, glycerophosphate choline, and seaweed oligosaccharide, with a mass ratio of (0.8-1.2):(0.3-0.6):(0.2-0.5); the anti-apoptotic active factor comprises low-concentration resveratrol, recombinant human epidermal growth factor, and glutathione, with a mass ratio of (0.1-0.3):(0.02-0.08):(0.4-0.7); the wet weight ratio of the composite bioactive protective system to the micro-lipid particles is controlled at 1:(120-180) to achieve micro-dose in-situ modification and avoid cytotoxicity caused by high concentrations of active factors.

[0012] The specific operational procedure for in-situ mild modification and simultaneous synergistic screening and enrichment includes: S1. Sterile adipose tissue is fed into the microparticle crushing chamber and subjected to low-shear cyclic crushing to obtain a crude micro-fat suspension. S2. The crude micro-fat suspension is pushed into the primary screening chamber by the low-pressure fluid in the chamber to remove large pieces of inert connective tissue impurities. S3. The suspension after primary screening continues to flow into the secondary screening and retention chamber. The micro-release module simultaneously starts the sustained-release composite bioactive protection system. The active factors are evenly infiltrated and retained micro-fat particles by circulating and permeating within the chamber, while simultaneously filtering out free oils, blood cells and waste fluid. After the simultaneous completion of S4, modification, screening and enrichment, the highly active micro-fat product with stem cell activity is collected under negative pressure in a sealed cavity. The cavity is not opened throughout the process to prevent external microbial contamination.

[0013] 8. The integrated method for screening, enriching, and modifying stem cell-rich micro-lipid components according to claim 7, characterized in that a circulating permeation guiding structure is provided inside the secondary screening and retention chamber, the guiding structure enabling the composite bioactive protection system to permeate bidirectionally along the micro-lipid particle accumulation layer in both the transverse and longitudinal directions, with a permeation circulation frequency of 3 to 6 times, ensuring that the gaps between each micro-lipid particle are infiltrated with active factors, eliminating the defects of uneven local modification and insufficient protection of surface particles.

[0014] This invention provides an integrated method for screening, enriching, and modifying the activity of micro-lipid components rich in stem cells. It offers the following advantages: 1. This invention overcomes the technical drawbacks of traditional micro-fat preparation processes, which involve step-by-step operations, multiple in vitro material transfers, and subsequent exogenous drug modification. It innovatively achieves the integrated and simultaneous completion of three core processes: fat micronization, impurity screening and enrichment, and in-situ activity modification. The entire process is carried out continuously in a closed, sterile device, eliminating the need for additional modification reagents and in vitro incubation. This significantly simplifies the preparation process, shortens the preparation time, and completely avoids the problems of material exposure and contamination, damage to adipose stem cells, apoptosis, and loss of active matrix caused by multiple transfers in traditional processes. It effectively improves the purity and production stability of micro-fat components, achieving efficient, sterile, and standardized mass production of high-quality stem cell micro-fat components.

[0015] 2. This invention utilizes a built-in micro-release module to precisely release moisturizing and anti-apoptotic compound active factors, enabling in-situ gentle modification of micro-fat particles. This allows the active factors to evenly penetrate the interparticle spaces, significantly improving the survival rate of adipose-derived stem cells and the preservation rate of the extracellular matrix. Compared to traditional micro-fat, which only has the function of filling and shaping, the micro-fat components prepared by this method have both excellent soft tissue filling effects and strong cell regeneration and collagen repair activities, effectively extending the survival period of transplanted fat and greatly expanding the application scenarios of the product. It can simultaneously meet the medical aesthetic and clinical repair needs such as filling depressions, repairing fine lines, improving skin texture, and repairing body surface wounds, greatly improving the therapeutic effect and clinical applicability of autologous micro-fat transplantation. Attached Figure Description

[0016] Figure 1 This is a flowchart illustrating the composition and flow of the active modification system of the present invention. Detailed Implementation

[0017] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] Please see the appendix Figure 1This invention provides an integrated method for screening, enriching, and modifying micro-fat components rich in stem cells. This method belongs to the field of autologous fat tissue engineering and regenerative medical aesthetics, and addresses the key challenges of existing step-by-step micro-fat preparation processes, such as fragmented procedures, significant cell damage, low stem cell survival rates, incomplete impurity removal, modification limited to the surface layer of particles, limited product functionality, and unstable clinical effects. The entire process relies on a dedicated, closed, aseptic integrated preparation device. This device is a fully enclosed aseptic chamber structure with no open operating windows, avoiding contamination from bacteria, fungi, and dust at the source. The device integrates three interconnected functional chambers: a microparticle fragmentation chamber, a gradient screening and retention chamber, and a micro-volume sustained-release modification chamber. These chambers are connected by precise flow-limiting channels, enabling closed-loop control of fluid flow rate, chamber pressure, and ambient temperature. The entire process requires no manual material handling or external placement. The device's built-in porous sustained-release microsphere-filled micro-release module is a detachable, precise drug release unit. The porous microspheres possess a nanoscale pore structure, enabling stable, uniform, and micro-level sustained release of bioactive factors, avoiding cell stimulation and toxic damage caused by instantaneous high-concentration release. The core innovation of this invention lies in transforming the three independent processes of traditional techniques—fat microparticle fragmentation, gradient screening and enrichment of inert impurities, and in-situ mild modification of microfat particles—into a synchronized, coordinated, and parallel integrated continuous process. This eliminates the need for post-preparation exogenous activity-protecting reagents and in vitro incubation modification, simplifying the process while maximizing the preservation of adipose-derived stem cell activity and extracellular matrix integrity. Specific systematic implementation steps and multiple parameter examples are as follows.

[0019] S1. Raw Material Pretreatment and Constant Temperature Aseptic Sealed Feeding: This invention selects autologous adipose tissue from the human waist, abdomen, inner thigh, or inner upper arm as raw material. These areas have high density of adipose-derived stem cells, good cell activity, and a low proportion of fibrous impurities, making them the optimal sampling sites. A low-negative-pressure aseptic aspiration device is used to extract the fat, with the aspiration negative pressure strictly controlled between -0.02 and -0.04 MPa to avoid large-scale rupture and necrosis of fat cells caused by high-negative-pressure aspiration. The extracted raw adipose tissue undergoes preliminary manual sorting to remove large blood clots, epidermal debris, fascia tissue, and obviously hardened fat blocks, retaining pure adipose tissue with a milky yellow color and uniform texture. Subsequently, the pretreated fresh autologous adipose tissue is rapidly and aseptically fed into the micronization and crushing chamber of the integrated preparation device. Immediately after feeding, the chamber's sealing structure is locked to form a completely sealed aseptic preparation environment, thoroughly preventing the intrusion of external air, microorganisms, and impurities. The synchronous start-up device's global constant temperature control system maintains the ambient temperature of the entire three-chamber interconnected system at a constant range of 32-36°C, which is within the physiologically mild range for mammals. The preferred implementation temperature of this invention is 34°C. This temperature range perfectly matches the survival and metabolic needs of adipose stem cells, avoiding both cell protein denaturation and stem cell apoptosis caused by high temperatures and cell metabolic stagnation and reduced activity caused by low temperatures, thus providing a stable cell survival microenvironment for the entire preparation process.

[0020] S2. Low-shear, gentle, homogeneous microparticle processing: This invention abandons the traditional high-speed, high-shear, and repeated violent crushing methods, and adopts a low-shear, reciprocating, flexible shearing structure specifically designed for the microparticle crushing chamber to achieve gentle microparticleization of adipose tissue. This invention provides three sets of gradient parameter examples to adapt to different textures of adipose tissue: Example 1: shear gap 100μm, single crushing time 5s, 6 cycles; Example 2: shear gap 200μm, single crushing time 8s, 4 cycles; Example 3: shear gap 300μm, single crushing time 12s, 3 cycles. All three examples achieve homogeneous crushing of adipose tissue, with the final micro-fat particles having a stable particle size concentrated in the range of 150–280μm, fully meeting the particle size standards for medical micro-fat injection and regeneration repair. The optimal example is Example 2, which is suitable for most human adipose tissue textures, exhibiting the best uniformity of micro-fat particle size after crushing, with no excessively crushed fine oil particles or large, uncrushed adipose tissue fragments. This step uses a low-intensity, low-frequency, flexible shearing method to completely preserve the intact capsule structure of the micro-fat particles, maximizing the protection of the adipose stem cells, vascular matrix components, and extracellular matrix fiber network nested inside the particles. It reduces mechanical damage from the source of microparticle formation and eliminates problems such as cell rupture, loss of contents, and stem cell inactivation caused by violent fragmentation in traditional processes.

[0021] S3. Two-stage gradient precise screening and enrichment and directional removal of inert impurities: The crude micro-fat suspension obtained by low-shear crushing is pushed at a constant speed by a sterile inert fluid inside a closed cavity, with the fluid flow rate controlled at 5 to 8 mL / s, so as to ensure the stable delivery of the suspension without particle extrusion and breakage, and then enters the gradient screening and retention cavity in sequence to complete two-stage fine screening and impurity removal. The first-stage coarse screen adopts a 400μm high-precision medical sterile screening mesh, which mainly intercepts and automatically discharges large connective tissues, coarse fat fiber bundles, sclerotic fat particles and residual fascia impurities through the directional sewage channel at the bottom, and only allows qualified micro-fat particles and the suspension phase to pass through, completing preliminary impurity removal. The second-stage fine screen adopts a 120μm ultra-fine screening mesh, which is the core enrichment and screening structure, and can precisely retain high-quality micro-fat components with a particle size of 120 to 300μm. The micro-fat particles in this particle size range have the characteristics of high stem cell density, intact extracellular matrix and high transplantation survival rate, and are the optimal components suitable for filling and regenerative repair. At the same time, through the negative pressure drainage system supporting the screening cavity, continuous negative pressure suction is performed to filter out invalid components with a particle size of less than 120μm, including free liquid inert oil, broken necrotic blood cells, tissue exudate waste liquid, broken cell debris and all other inert impurities without biological activity. Through the combined structure of two-stage gradient screening, the present invention realizes the layered treatment of "coarse impurity removal + fine enrichment", thoroughly solves the defects of traditional static precipitation and single-layer filtration processes such as incomplete impurity removal, waste of active components, and mixing of large and small particles, and greatly improves the purity, uniformity and overall activity quality of the finished micro-fat.

[0022] S4. Simultaneous Screening, Enrichment, and In-situ Micro-release Active Modification: The core innovation of this invention lies in the complete synchronization and parallel operation of the two-stage gradient screening and enrichment process with the in-situ active modification process, without time lag or process intervals. Micro-fat particles are immediately infiltrated and protected by active factors while being enriched, eliminating the attenuation of stem cell activity caused by waiting outside the body. The device's built-in micro-release module is pre-activated, and the release rate is dynamically matched based on the wet weight of the micro-fat particles. The module is pre-loaded with a composite bioactive protection system, which consists of two main components: a moisturizing and protective film component and an anti-apoptotic and regenerative component, each with its own function and synergistic effect. The moisturizing active factor is composed of hyaluronic acid oligosaccharide, glycerophosphate choline, and seaweed oligosaccharide, with an optimal mass ratio of 1:0.4:0.3. This ratio forms a uniform moisturizing protective film on the surface and between the micro-lipid particles, locking in cell moisture, maintaining cell osmotic pressure balance, and preventing particle dehydration, inactivation, and structural collapse. The anti-apoptotic active factor is composed of resveratrol, recombinant human epidermal growth factor, and glutathione, with an optimal mass ratio of 0.2:0.05:0.5. This compound system can effectively scavenge oxidative free radicals generated during preparation, inhibit stem cell apoptosis pathways, and mildly activate cell repair and regeneration potential. The optimal feed ratio of the composite bioactive protective system to the wet weight of the micro-lipid particles is 1:150. This micro-dose mode of action can achieve highly efficient activity protection while completely avoiding the cytotoxicity and overstimulation side effects caused by high concentrations of biological factors. Controls were implemented within a specified ratio range. Fine adjustments within the range defined in the claims achieved excellent modification effects, adapting to different preparation scenarios with varying activity requirements.

[0023] During the sustained release of active factors, the secondary sieving and retention chamber activates a full-area circulating permeation and guiding structure, constructing a three-dimensional circulating wetting system of transverse flat permeation and longitudinal penetrating permeation. This completely changes the shortcomings of traditional modification methods that can only wet the surface of particles. The present invention preferably uses four cycles of permeation, with each cycle lasting 15 seconds, ensuring that the composite active factor solution can fully penetrate into the interstices and core structure of the micro-fat particles, achieving comprehensive and uniform modification of each micro-fat particle without dead angles. The entire modification and screening process maintains a slightly positive pressure and mild environment within the chamber, with the pressure stable at 0.01–0.02 MPa. There is no high-speed fluid impact, no mechanical extrusion, and no air exposure, maintaining a stable microenvironment for stem cell survival throughout the process. This achieves a high degree of synergistic integration of the three processes of microparticle formation, screening and enrichment, and activity modification, solving the core problems of activity loss, uneven modification, and unstable quality in traditional step-by-step processes from a technological mechanism perspective.

[0024] S5. Aseptic and closed collection of finished products: After all screening, impurity removal and in-situ activity modification processes are completed, the modified high-activity micro-fat products are collected through the built-in negative pressure collection pipeline in a completely closed and sterile state. High-quality stem cell micro-fat components that can be used for clinical filling, skin repair and wound healing are obtained directly without the need for secondary purification and secondary modification. The entire process is completed in a continuous closed loop.

[0025] To fully and objectively verify the technical superiority of the integrated process of this invention compared with traditional processes, this invention sets up three parallel examples and one comparative example of traditional step-by-step preparation. Parallel control experiments were conducted under the premise of uniform material sources, uniform sterile laboratory environment, uniform temperature and humidity conditions, and uniform fat feeding weight. The results of various performance indicators are as follows: The micro-fat fraction obtained by the traditional step-by-step preparation process (centrifugation purification + open filtration + subsequent exogenous drug incubation) had an average adipose stem cell survival rate of only 58%, an extracellular matrix integrity retention rate of only 75%, and a high residual rate of inert impurities and broken tissue. The original method had a low particle size distribution (8.2%), poor particle size uniformity, severe mixing of large and small particles, high early absorption rate after transplantation, and only basic physical filling effect without significant collagen regeneration and skin repair functions. In the three gradient examples of this invention, the micro-fat component prepared in the optimal example 2 achieved an average adipose stem cell survival rate of 93%, an extracellular matrix integrity retention rate of 95%, an inert impurity residue rate of less than 1%, and a particle size uniformity >96%. The other two gradient examples showed stem cell survival rates of no less than 88% and extracellular matrix retention rates of no less than 90%, with all core performance indicators significantly superior to traditional processes. Testing revealed that the micro-fat component prepared in this invention significantly reduced oxidative stress levels, substantially decreased apoptosis rates, and significantly increased expression levels of active regeneration factors, exhibiting excellent biological activity and stability.

[0026] Through multiple batches of in vitro testing and clinical pilot applications, the highly active stem cell micro-fat component prepared by the integrated method of this invention possesses dual core efficacy advantages. On the one hand, the product has uniform particles, high purity, and is free of harmful impurities. After transplantation, it results in high soft tissue filling smoothness, natural shaping effect, and significantly improved long-term fat retention rate, greatly reducing the risk of complications such as unevenness, rapid absorption, and nodule hyperplasia after traditional fat transplantation. On the other hand, thanks to the preservation of highly active adipose stem cells and intact extracellular matrix, the modified micro-fat can continuously secrete a variety of regenerative active factors at the transplantation site, effectively activating fibroblast proliferation, promoting dermal collagen regeneration, and repairing the skin barrier structure. It can specifically improve problems such as facial static fine lines, enlarged pores, dull skin texture, and photoaging damage. At the same time, it can be applied to the repair and healing of superficial skin wounds, minimally invasive postoperative wounds, and small soft tissue defects, effectively shortening the wound healing cycle and improving the smoothness of the repaired skin. This invention completely breaks through the technical limitations of traditional micro-fat, which can only physically fill and has no regenerative and repair activity. It realizes the technical upgrade of micro-fat materials from simple filling consumables to regenerative and repair active materials, greatly expanding the clinical application scenarios and therapeutic value of autologous micro-fat.

[0027] 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 alterations 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. A method integrating screening, enrichment, and activity modification of micro-lipid components rich in stem cells, characterized in that, The process includes the following synchronous and coordinated steps: micronization and fragmentation of adipose tissue, gradient screening and enrichment of adipose impurities, and in-situ active and mild modification. The entire process is completed continuously inside a closed and sterile preparation device, with no material transfer or in vitro static transition between steps. The bioactive protection system required for modification is supplied in situ through the built-in micro-release module of the device, eliminating the need for additional exogenous active reagents after preparation. During the in-situ active and mild modification process, the micro-release module precisely releases moisturizing and anti-apoptotic active factors adapted to the micro-fat particles. The two types of active factors penetrate evenly into the interstitial space of the micro-fat particles, simultaneously completing the retention and enrichment of high-quality micro-fat components rich in adipose stem cells and extracellular matrix, while removing inert oil impurities, connective tissue fragments, and inert particles of blood cell sediment that have no regenerative activity.

2. The integrated method for screening, enriching, and modifying the activity of micro-lipid components rich in stem cells according to claim 1, characterized in that, The closed aseptic preparation device integrates at least a microparticle fragmentation chamber, a gradient screening and retention chamber, and a micro-volume slow-release modification chamber, which are connected in a three-chamber structure. The air pressure, temperature, and fluid flow rate inside the three chambers are linked and regulated to maintain a mild physiological environment of 32-36°C throughout the process, avoiding stem cell damage and apoptosis caused by high temperature and high speed shearing.

3. The integrated method for screening, enriching, and modifying the activity of micro-lipid components rich in stem cells according to claim 2, characterized in that, The adipose tissue microparticle fragmentation process employs a low-shear reciprocating shear fragmentation structure with a shear gap controlled at 100–300 μm and a single fragmentation cycle duration of 5–12 s. The fragmented output micro-fat particles are concentrated in the range of 150–280 μm in size, ensuring the complete preservation of adipose stem cells and extracellular matrix within the particles and preventing cell membrane rupture.

4. The integrated method for screening, enriching, and modifying the activity of micro-lipid components rich in stem cells according to claim 3, characterized in that, The gradient screening and enrichment process adopts a two-stage gradient interception sieving structure; the first-stage sieving has a pore size of 400μm, which intercepts large connective tissue, fatty fiber, and inert impurities and discharges them in a directional manner; The secondary sieve has a pore size of 120μm, which can retain and enrich micro-fatty components with a particle size of 120-300μm and high stem cell density. Free oil with a particle size of less than 120μm and broken blood cell waste liquid are discharged under negative pressure at the same time, realizing the real-time separation of high-quality active components and inert impurities.

5. The integrated method for screening, enriching, and modifying the activity of micro-lipid components rich in stem cells according to claim 4, characterized in that, The device has a built-in micro-release module, which is a porous microsphere-filled sustained-release unit. The sustained-release microspheres are pre-loaded with a composite bioactive protection system. The composite bioactive protection system is composed of moisturizing active components and anti-apoptotic active components. The distribution ratio of each component in the system is dynamically matched to the release rate based on the cell density of the micro-lipid particles obtained by secondary sieving.

6. The integrated method for screening, enriching, and modifying the activity of micro-lipid components rich in stem cells according to claim 5, characterized in that, The moisturizing active factor comprises hyaluronic acid oligosaccharide, glycerophosphate choline, and seaweed oligosaccharide, with a mass ratio of (0.8–1.2):(0.3–0.6):(0.2–0.5); the anti-apoptotic active factor comprises low-concentration resveratrol, recombinant human epidermal growth factor, and glutathione, with a mass ratio of (0.1–0.3):(0.02–0.08):(0.4–0.7); the wet weight ratio of the composite bioactive protective system to the micro-lipid particles is controlled at 1:(120–180), achieving micro-dose in-situ modification and avoiding cytotoxicity caused by high concentrations of active factors.

7. The integrated method for screening, enriching, and modifying micro-lipid components rich in stem cells according to claim 6, characterized in that, The specific operational procedure for in-situ mild modification and simultaneous synergistic screening and enrichment includes: S1. Sterile adipose tissue is fed into the microparticle crushing chamber and subjected to low-shear cyclic crushing to obtain a crude micro-fat suspension. S2. The crude micro-fat suspension is pushed into the primary screening chamber by the low-pressure fluid in the chamber to remove large pieces of inert connective tissue impurities. S3. The suspension after primary screening continues to flow into the secondary screening and retention chamber. The micro-release module simultaneously starts the sustained-release composite bioactive protection system. The active factors are evenly infiltrated and retained micro-fat particles by circulating and permeating within the chamber, while simultaneously filtering out free oils, blood cells and waste fluid. After the simultaneous completion of S4, modification, screening and enrichment, the highly active micro-fat product with stem cell activity is collected under negative pressure in a sealed cavity. The cavity is not opened throughout the process to prevent external microbial contamination.

8. The integrated method for screening, enriching, and modifying the activity of micro-lipid components rich in stem cells according to claim 7, characterized in that, The secondary screening and retention chamber is equipped with a circulating permeation guiding structure. The guiding structure allows the composite bioactive protection system to permeate in both the transverse and longitudinal directions along the micro-fat particle accumulation layer. The permeation circulation frequency is 3 to 6 times, ensuring that the gaps between each micro-fat particle are infiltrated with active factors, eliminating the defects of uneven local modification and insufficient protection of surface particles.

Images