Dynamic culture system and culture method for acellular scaffold tissue

Abstract

The invention belongs to the technical field of tissue engineering scaffold in-vitro culture, and particularly relates to a dynamic culture system and culture method for acellular scaffold tissue. The dynamic culture system comprises: a housing in communication with an oxygen supply device; the culture dishes are arranged on the upper portion of the shell in a stacked mode, and two connectors are formed in the bottom wall of each culture dish; the perfusion system comprises an oxygenator and a peristaltic pump which are communicated in sequence, and a polytetrafluoroethylene hollow fiber pipe in the oxygenator and the peristaltic pump are respectively connected with one interface; the joint is positioned in the culture dish and is communicated with the interface connected with the oxygenator; the power supply is used for supplying power to the peristaltic pump; the controller is used for controlling the operation of the peristaltic pump. The dynamic culture system effectively supports three-dimensional tissue growth, and overcomes the defects of a traditional system. The device is compact in design, simple and convenient to operate and capable of maintaining complex tissue constructs, so that the device becomes a powerful tool for tissue engineering application and regenerative medicine research.

Description

Technical Field

[0001] The present invention belongs to the technical field of in vitro culture of tissue engineering scaffolds, and particularly relates to a dynamic culture system and a culture method for acellular scaffold tissues. Background Art

[0002] Organ transplantation remains the most effective method for treating various end-stage diseases, but its application is limited by the severe shortage of donor organs. In the past decade, acellular organ scaffolds have emerged as key materials in tissue engineering, showing great potential for constructing engineered organs. By removing the cellular components from donor tissues while completely retaining the extracellular matrix (ECM) and vascular structures, these scaffolds provide an optimal microenvironment for cell adhesion, growth, and differentiation. Therefore, acellular scaffolds have been widely used in the construction of various tissue-engineered organs, including the heart, liver, kidney, blood vessels, and lungs, providing a feasible solution for organ replacement and regenerative medicine. Despite their great potential, constructing tissue-engineered organs using acellular scaffolds still faces major challenges, mainly due to the complexity, inefficiency, and lack of flexibility of existing methods. Existing culture systems (especially organoid research) usually rely on the complex integration of multiple external components, such as peristaltic pumps, adapters, tubing, and reservoirs. These systems are cumbersome to operate, rely on highly skilled personnel, have a high failure rate, and result in serious cell loss. The complexity of the system and multi-step operations further increase the operation error and the risk of contamination. In addition, the tissue culture process is time-consuming and often involves expensive or scarce cell sources, making experimental losses unbearable. To address these problems, there is an urgent need to develop a more adaptable and user-friendly dynamic culture platform to efficiently and reliably support the construction of complex three-dimensional in vitro models.

[0003] In the past decade, researchers have explored various methods to simplify and optimize the three-dimensional culture of acellular scaffolds, such as converting the scaffolds into hydrogels, homogenates, pump-free systems, or using microfluidic chip technology. Although these methods have improved some ease of use, they have all failed to address a key limitation: the diffusion barrier, that is, oxygen and nutrients in liquids (blood, culture medium, tissue fluid) can only freely diffuse into the tissue interior up to 200 μm and cannot exceed this distance. Therefore, how to simulate the effective vascular perfusion of blood flow to enable the liquid to penetrate deep into the tissue interior remains a technical problem to be solved. Summary of the Invention

[0004] To address the challenges of dynamic culture, the present invention proposes a dynamic culture system for acellular scaffold tissues, whose design focuses on high miniaturization, reducing tubing connections, ensuring easy observation and assembly, and maintaining low cost, thereby providing a practical and efficient solution for simulating the effective vascular perfusion of blood flow and constructing three-dimensional tissue-engineered organs. The present invention first provides a dynamic culture system for acellular scaffold tissues, comprising:

[0005] A housing, on the side wall of which an oxygen pipeline is provided; A culture dish, which is stacked above the housing, and two interfaces are provided on the bottom wall of the culture dish; A perfusion system, which is located inside the housing, the perfusion system includes an oxygenator and a peristaltic pump, the oxygenator is composed of an aeration housing and a polytetrafluoroethylene hollow fiber tube located inside the aeration housing, the surface of the polytetrafluoroethylene hollow fiber tube has a pore structure, one end of the polytetrafluoroethylene hollow fiber tube is connected to one of the interfaces, the other end is connected to the peristaltic pump, and the peristaltic pump is also connected to the other interface; the aeration housing is communicated with the oxygen pipeline, and the oxygen pipeline is communicated with an oxygen supply device; A connector, which is located inside the culture dish, the connector is communicated with the interface connected to the peristaltic pump, and the connector is used for connecting a decellularized scaffold tissue placed in the culture dish; A power supply, which is electrically connected to the peristaltic pump and is used to provide power supply for the peristaltic pump; A controller for controlling the operation of the peristaltic pump.

[0006] In view of the deficiencies of the prior art, the present invention has developed a new type of miniaturized dynamic culture system. This system has the advantages of simplified assembly and reduced dead space volume. Despite recent progress in the field of tissue engineering, traditional 3D culture methods using decellularized scaffolds still have key defects. Current methods generally process decellularized scaffolds into hydrogels, bioinks or homogenates, which, although improving operability, destroy the most valuable property of the scaffold - the natural three-dimensional vascular structure. The vascular network is crucial for the nutrient delivery and waste removal of natural tissues, and the processed scaffold materials cannot reproduce this structure, resulting in tissue growth being limited by diffusion, and usually the thickness of the construct does not exceed 200 μm. In addition, processing the scaffold into a hydrogel will significantly change the biochemical and biomechanical properties of the extracellular matrix (ECM), including the structural arrangement, mechanical properties and spatial distribution of bioactive molecules, which may affect cell behavior and tissue development. Using the dynamic culture system of the present invention can preserve the integrity of the natural vascular network and provide continuous perfusion, solve these limitations, and support the development of larger and more physiologically relevant tissue constructs. Taking the decellularized liver scaffold as a proof-of-concept model, we have demonstrated that this dynamic culture system can significantly improve the dynamic culture efficiency while maintaining excellent cell viability, proliferation ability and functional expression. The research results show that this user-friendly system is expected to become a general tool for constructing various tissue engineering organs and promoting regenerative medicine research.

[0007] Further, the oxygen supply device is an oxygen cylinder with a flow meter or an oxygen generator.

[0008] Further, the polytetrafluoroethylene hollow fiber tube is spiral or wavy.

[0009] Furthermore, the inner diameter of the polytetrafluoroethylene hollow fiber tube is 0.9 - 1 mm, the outer diameter is 1.6 - 1.7 mm, the porosity exceeds 60%, the pore size ranges from 0.2 - 50 μm, and the length is 1.8 - 2 m.

[0010] Furthermore, the top of the aeration housing is semi-closed.

[0011] Secondly, the present invention provides a method for dynamically culturing acellular scaffold tissues, which is carried out using the above-mentioned dynamic culture system. The culture method includes: S1. Prepare acellular scaffold tissues; S2. Completely perfuse the medium containing the corresponding cells of the tissue into the acellular scaffold tissues in advance; S3. Insert a silicone tube into the blood circulation inlet of the acellular scaffold tissues, and then connect it to the connector; S4. Place the dynamic culture system in a CO 2 incubator and statically culture for 4 - 5 h to promote cell adhesion; S5. Start the peristaltic pump for continuous perfusion, synchronously supplement oxygen, and change the medium daily.

[0012] Furthermore, the flow rate of the peristaltic pump is set to 1.5 - 2 mL / min.

[0013] Furthermore, the volume concentration of the supplemented oxygen gas is 88% - 90%, and the flow rate is 20 - 25 mL / min.

[0014] Compared with the prior art, the present invention has the following beneficial effects: In the present invention, we developed and validated a novel miniaturized dynamic culture system, which solved several key limitations of traditional three-dimensional (3D) culture systems. This dynamic culture system integrates core components such as pumps, oxygenators, and perfusion systems into a compact and user-friendly design, significantly reducing system complexity while maintaining optimal culture conditions. Its innovative features include: integrated sterilization ability, minimum dead volume (3 mL), rapid assembly time (<5 min), and efficient gas exchange through a polytetrafluoroethylene (PTFE) membrane. Experiments using acellular liver scaffolds showed that this dynamic culture system can effectively support cell growth and tissue-specific functions.

[0015] The dynamic culture system developed in this invention has multiple significant advantages compared to traditional systems. First, its innovative design prioritizes simplicity and operational efficiency while maintaining optimal culture conditions. The integrated housing manufactured by 3D printing can be sterilized as a whole, significantly reducing the contamination risk during the assembly process. The streamlined design and compact size enable single-person operation to complete the assembly within 5 minutes, greatly improving the operational efficiency compared to traditional systems that often require multi-person collaboration and complex setup procedures. Different from traditional platforms that usually occupy the entire incubator, our miniaturized system can coexist with standard culture dishes and flasks, maximizing the utilization of incubator space. This space efficiency and miniaturization feature enable it to be combined into an expandable cell culture factory, supporting efficient small-batch tissue production. Second, the system demonstrates significant cost-effectiveness through the use of standard laboratory materials and efficient design. The perfusion circuit and oxygenator only require a pre-charge volume of 2.5 mL, significantly reducing the dead volume compared to traditional systems, while maintaining optimal culture conditions with the use of standard culture medium volume. Third, the intelligent remote monitoring and control functions achieved through a mobile application improve user-friendliness, supporting real-time system monitoring and rapid intervention. In addition, the biological verification of culturing AML12 cells based on a decellularized liver scaffold shows that this platform can promote cell proliferation and maintain hepatocyte function, which is confirmed by comprehensive analysis results. These results together prove that this platform is a highly promising tool in dynamic 3D culture applications.

[0016] Sufficient oxygenation is crucial for the success of three-dimensional tissue culture, but existing oxygenation methods have significant limitations. Traditional direct bubble oxygenation can increase the oxygen content, but it introduces bubbles into the tissue construct and generates excessive foam in the culture medium, leading to an increased risk of contamination and culture failure. Commercial membrane oxygenators, although a commonly used solution, have high system complexity, large pre-charge volume, high cost, and a scarcity of miniaturized versions. They often require the modification of large equipment, which may compromise sterility and functionality. Previous attempts to use silicone tubes as an alternative oxygenation method were limited due to insufficient gas exchange rate. To address these problems, our system uses a PTFE membrane, which combines water and air permeability, excellent gas permeability, high exchange rate, biocompatibility, and stability. Experiments show that the partial pressure of oxygen in the culture medium and diluted blood samples increases significantly after being treated with the PTFE membrane. The significant improvement in oxygen transfer efficiency, combined with the low cost and easy integration advantages of this membrane, marks a major advancement in tissue culture oxygenation technology. This innovation provides reliable oxygen supply while maintaining the simplicity and sterility of the system, providing a key guarantee for long-term three-dimensional tissue culture.

[0017] The application potential of this dynamic culture system far exceeds the field of acellular scaffolds. Although we have verified its efficacy with acellular liver scaffolds, this system can be flexibly adapted to various tissue engineering requirements. In organ engineering, its adjustable flow rate parameters and efficient oxygenation system can support the culture of acellular scaffolds such as the heart, kidney, lung, and blood vessels. In addition to acellular scaffolds, this platform can also be used for three-dimensional systems such as synthetic polymer scaffolds, hydrogel constructs, and organoid culture, and can be used as a tool for studying tissue development, disease modeling, and cell-matrix interactions under dynamic conditions. Its compatibility with standard imaging techniques and potential for real-time monitoring further enhance its application value in basic research and high-throughput drug screening. The scalability and cost-effectiveness of the design also make it an ideal choice for drug development and toxicology testing. Brief Description of the Drawings

[0018] Figure 1 Schematic diagram of the results of the dynamic culture system for acellular scaffold tissues, where 1. outer shell, 2. culture dish, 3. oxygen pipeline, 4. peristaltic pump, 5. aeration housing, 6. polytetrafluoroethylene hollow fiber tube, 7. power supply.

[0019] Figure 2 Schematic design diagram of the dynamic culture system and culture method for acellular scaffold tissues.

[0020] Figure 3 Schematic diagram of the structure of the dynamic culture system for acellular scaffold tissues and the realization of dynamic culture, where: A is a schematic diagram of the realization of dynamic culture of acellular scaffold tissues.

[0021] B is an exploded view of the dynamic culture system and the user interface of the mobile control application.

[0022] C is the process of cell seeding using an infusion pump, and cells are controllably delivered into the acellular liver scaffold through the portal vein (injection rate: 1 mL / min).

[0023] D is a representative image of the dynamic culture system. Left figure: the assembled platform in operation; right figure: a close-up view of the acellular liver scaffold under perfusion culture conditions.

[0024] Figure 4 For the oxygenation efficiency and perfusion pressure characteristics of the dynamic culture system, where: A is a morphological comparison of the polytetrafluoroethylene hollow fiber tube in the oxygenator of the present invention and a commercial oxygenator.

[0025] B is a comparison of the oxygenation performance of the oxygenator of the present invention and a commercial oxygenator.

[0026] C is the partial pressure of oxygen before and after oxygenation of different media.

[0027] D represents the change of perfusion pressure with perfusion flow rate.

[0028] Figure 5 is the characterization of the decellularized liver scaffold, where: A is the liver decellularization process, including freeze-thaw cycles, deionized water washing, sodium dodecyl sulfate (SDS), Triton X-100, phosphate buffered saline (PBS) rinsing, and cryopreservation.

[0029] B are representative images of the liver at different stages of decellularization, showing the gradual removal of cellular components while maintaining the vascular structure.

[0030] C is a scanning electron microscope (SEM) image of the decellularized liver scaffold, showing that the extracellular matrix structure is maintained. Scale bar: 10 μm.

[0031] D is a hematoxylin and eosin staining of the decellularized scaffold.

[0032] E is a Masson's trichrome staining of the decellularized scaffold.

[0033] F is an Alcian blue staining of the decellularized scaffold.

[0034] G is an immunofluorescence staining of type I collagen.

[0035] H is an immunofluorescence staining of type IV collagen.

[0036] I is an immunofluorescence staining of fibronectin.

[0037] J is a statistical graph of DNA quantification of native liver and decellularized liver scaffold (DLM).

[0038] Figure 6 is the comparison between dynamic culture and static culture in terms of supporting cell proliferation and viability, where: A are the macroscopic morphological changes of the decellularized liver scaffold during 7 days of dynamic culture (top) and static culture (bottom). Scale bar: 2 cm.

[0039] B is a hematoxylin and eosin (H&E) staining. In the scaffold of dynamic culture, cells attach firmly and are well-distributed, while the static culture sample shows karyolysis and morphological damage. The * in the figure represents the vascular lumen. Scale bar: 50 μm.

[0040] C is a Ki67 immunofluorescence staining. Compared with static culture, cell proliferation in the scaffold of dynamic culture is significantly higher. Scale bar: 50 μm.

[0041] D is a TUNEL staining. Compared with static culture, the apoptosis rate in the constructs of dynamic culture is significantly reduced. Scale bar: 50 μm.

[0042] E is the staining of albumin (ALB). Compared with the static control group, the expression of albumin in the dynamic culture group was significantly higher. Scale bar: 50 μm.

[0043] Figure 7 Quantitative analysis results of Ki67-positive cells, TUNEL-positive cells, and ALB-positive cells in decellularized liver scaffolds under dynamic and static culture conditions. A shows the relative level of Ki67-positive cells, B shows the relative level of TUNEL-positive cells, and C shows the relative level of ALB-positive cells.

[0044] Figure 8 Daily and cumulative urea secretion curves of decellularized liver scaffolds under dynamic and static culture conditions. Detailed implementation manners

[0045] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well-known to those skilled in the art. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0046] Example 1: Construction and performance characterization of a dynamic culture system for decellularized scaffold tissues 1. Method 1.1 Construction of a dynamic culture system for decellularized scaffold tissues As Figure 3 shown, the dynamic culture system for decellularized scaffold tissues in this example includes: A 3D-printed outer shell 1, compatible with ethylene oxide sterilization, with an oxygen pipeline 3 provided on its side wall. An oxygen supply device is connected to the oxygen pipeline 3. In this example, the oxygen supply device is an oxygen cylinder, and the oxygen cylinder is equipped with a flow meter that can control the flow rate, and the flow rate of oxygen is controlled by the flow meter.

[0047] A culture dish 2, which is stacked above the outer shell 1, and two interfaces are provided on the bottom wall of the culture dish 2; Perfusion system, located within the housing 1, the perfusion system includes an oxygenator and a peristaltic pump 4, the peristaltic pump 4 is driven by an N20 reduction motor to achieve precise circulation of the culture medium. The oxygenator consists of an aeration housing 5 and a spiral-shaped polytetrafluoroethylene hollow fiber tube 6 located within the aeration housing 5. One end of the polytetrafluoroethylene hollow fiber tube 6 is connected to one of the interfaces, and the other end is connected to the peristaltic pump 4. The peristaltic pump 4 is also connected to another interface; the aeration housing 5 is in communication with the oxygen pipeline 3; the surface of the polytetrafluoroethylene hollow fiber tube 6 has a pore structure, with an inner diameter of 0.9 mm and an outer diameter of 1.7 mm. It has selective gas permeability and chemical stability, with a pore size range of 0.2 - 50 μm, a porosity exceeding 60%, and a length of 2 m. The spiral shape allows the relatively long polytetrafluoroethylene hollow fiber tube 6 to be accommodated within the aeration housing 5, and can enable oxygen to preferentially fill the aeration housing 5 and come into contact with the polytetrafluoroethylene hollow fiber tube 6, improving the efficiency of oxygen exchange; the top of the aeration housing 5 is semi-closed, and there are 2 mm small holes on the side wall of the housing 1 to prevent excessive pressure.

[0048] Connector, located within the petri dish 2, the connector is in communication with the interface connecting the peristaltic pump 4, and the connector is used to connect the decellularized scaffold tissue placed within the petri dish 2.

[0049] Power supply 7, electrically connected to the peristaltic pump 4, used to provide power supply for the peristaltic pump 4, and the power supply 7 is arranged on the side wall of the housing 1; Controller for controlling the operation of the peristaltic pump 4, using ESP8266 as the main control chip, having WiFi function, enabling remote control and data transmission, docking with the Blinker Internet of Things platform, supporting real-time monitoring and adjustment of perfusion parameters by mobile applications; controlling the rotation speed and direction of the peristaltic pump 4 through PWM signals to achieve controllable flow rate, connecting the control signal input terminal of the peristaltic pump 4 to the GPIO pin of ESP8266, and connecting the positive and negative poles to the power supply 7. Since this circuit design is a conventional technology, it will not be elaborated here.

[0050] 1.2 Performance characterization of the dynamic culture system for decellularized scaffold tissue in this embodiment: (1) Oxygen partial pressure test: Using an ABL800 FLEX blood gas analyzer (Radiometer, Denmark) and an OxyLite ix408 oxygen partial pressure monitoring system (Oxford Optronix Ltd., UK), compare the differences in oxygen partial pressure between the oxygenator of the present invention and commercial oxygenators in water, PBS, culture medium, and 5% diluted blood. (2) Hydraulic performance test: Using a calibrated pressure sensor (Taimeng Technology, Chengdu, China) to measure the perfusion pressure at different flow rates.

[0051] 2. Results 2.1 Design and system parameters The dynamic culture system ensures optimal performance and compactness through precision engineering. The outer shell is designed using SolidWorks and manufactured by LCD stereolithography 3D printing, presenting a cylindrical structure (10 cm in diameter and 3 cm in height). The peristaltic pump is designed to have an adjustable flow rate (0.5 - 3 mL / min). The oxygenator, peristaltic pump, and decellularized scaffold are connected by medical-grade silicone tubing (inner diameter 0.8 mm, outer diameter 1.9 mm), with only an initial volume of 2.6 mL of culture medium required, significantly reducing reagent consumption compared to traditional systems.

[0052] 2.2 Oxygenation Capacity and Dynamic Perfusion The core feature of the system is its highly efficient oxygenator. The oxygenation circuit is a 2.0-meter-long polytetrafluoroethylene hollow fiber tube ( Figure 4 A), with an effective gas exchange area of approximately 6000 mm². The custom-designed oxygenator has a significantly smaller volume than commercial membrane oxygenators. Although its oxygenation capacity is lower than that of commercial devices, it can still meet the oxygen requirements for cell culture ( Figure 4 B). To verify the oxygenation efficiency, the system was tested for changes in partial pressure of oxygen in various perfusion media (water, PBS, culture medium, and 5% diluted blood). The partial pressure of oxygen in all media increased significantly after passing through the oxygenator of the present invention, with the value approximately doubling compared to before oxygenation ( Figure 4 C). In addition to oxygenation, optimizing perfusion parameters is crucial for maintaining cell viability and preventing mechanical damage. The perfusion pressure is positively correlated with the flow rate ( Figure 4 D). Since only the middle lobe of the liver scaffold was used in the experiment, it was verified that a flow rate of 1.5 - 2.0 mL / min can achieve sufficient perfusion while maintaining an appropriate pressure (preventing cell detachment). This flow rate range was subsequently used for all dynamic culture experiments.

[0053] Example 2: Dynamic Culture of Decellularized Scaffold Tissue 1. Method 1.1 Liver Acquisition and Preservation All animal experiments were approved by the Animal Ethics Committee of Xi'an Jiaotong University and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals. Adult male Sprague - Dawley rats (250 - 300 g, n = 10) were used as liver donors. Before the operation, the rats had free access to food and water. After anesthesia with isoflurane (2%), the rats were heparinized systemically via the penile vein (1000 U) to prevent blood clotting. The abdomen was opened to expose the portal vein, and an 18G intravenous catheter was inserted. The liver was perfused with normal saline (4 mL / min, 5 min) at room temperature until the outflow was clear. After sacrifice, the surrounding tissues of the liver were carefully dissected while preserving the vascular structure, and the liver was immediately stored at -80 °C until decellularization treatment.

[0054] 1.2 Preparation of Decellularized Liver Scaffold The cryopreserved liver was subjected to three freeze-thaw cycles from -80 °C to 37 °C to lyse the cells. The decellularization process was completed by perfusion through the portal vein, and a peristaltic pump maintained a constant flow rate of 4 mL / min to achieve liver decellularization. The specific procedure is as follows:

[0055] (1)Irrigate with deionized water for 1 h; (2)Perfuse with 0.1% sodium dodecyl sulfate (SDS, MP Biomedicals, USA) for 4 h to remove cellular components; (3)Treat with 1% Triton X-100 for 2 h; (4)Rinse with phosphate-buffered saline (PBS) for 4 h to remove residual detergents; (5)Soak in 0.1% (v / v) peracetic acid for 24 h for sterilization, followed by neutralization with PBS. The treated scaffolds were used immediately or stored in PBS containing 5% penicillin-streptomycin (PS) at 4 °C.

[0056] 1.3 Dynamic culture to achieve recellularization AML12 cells were expanded in DMEM / F12 medium (cell concentration of 1×10 7 / ml), and then injected into the decellularized liver scaffolds through the portal vein at a rate of 1 mL / min ( Figure 3 C), with a single injection volume of 1 mL, completed in 3 times. The inoculated scaffolds were connected to the dynamic culture system through a stainless-steel adapter, and the system was pre-filled with medium to ensure complete perfusion. The assembled constructs were placed in a CO 2 incubator (continuously providing 5% CO 2 , to stabilize the pH of the medium) for an initial static culture for 4 h to promote cell adhesion. Subsequently, continuous perfusion was initiated (the scaffolds of the liver are relatively large, and the flow rate was set at 2 mL / min. If culturing scaffolds of other smaller tissues, such as the spleen and kidney, the flow rate was set at 1.5 mL / min), and oxygen with a volume concentration of 90% was supplemented synchronously (flow rate of 25 mL / min). 30 mL of medium was changed daily and 2 mL of samples were collected for analysis, and samples were taken after 7 days of dynamic culture.

[0057] The static control group adopted the same cell inoculation procedure, but was placed in a standard culture dish for non-perfusion culture. The culture conditions, medium change frequency, sampling time, and experimental period were the same as those of the dynamic group.

[0058] 1.4 Histological analysis Samples from dynamic and static cultures were taken on the 7th day for histological evaluation: Hematoxylin-eosin (H&E) staining: Observe tissue morphology and cell distribution; Ki67 immunofluorescence staining: Label proliferating cells; TUNEL assay (Terminal - deoxynucleotidyl Transferase Mediated Nick End Labeling): To detect apoptotic cells.

[0059] 1.5 Liver function assessment Collect medium samples daily for biochemical analysis: Albumin (ALB) secretion: Evaluated by immunofluorescence staining; Urea production: Detected using a commercial kit (Nanjing Jiancheng, China) based on urease colorimetry. All experiments were repeated three times.

[0060] 2. Results 2.1 Characterization of decellularized liver scaffolds Sequential decellularization treatment ( Figure 5 A) effectively removed cellular components and retained the natural extracellular structure. During the decellularization process, the liver tissue gradually changed from its characteristic brown color to a transparent state while retaining a clear vascular structure ( Figure 5 B). Through comprehensive morphological analysis by scanning electron microscopy (SEM) ( Figure 5 C), hematoxylin - eosin (H&E) staining ( Figure 5 D), Masson's trichrome staining ( Figure 5 E) and alcian blue staining ( Figure 5 F), it was confirmed that cellular and nuclear components were completely removed, and the three - dimensional ultrastructure and extracellular matrix (ECM) components were intactly retained. Immunofluorescence analysis showed the retention of key ECM proteins: type I collagen ( Figure 5 G), type IV collagen ( Figure 5 H) and fibronectin ( Figure 5 I) staining confirmed that both the structural framework components and the vascular basement membrane components were retained. Quantitative DNA analysis of the decellularized liver scaffolds ( Figure 5 J) further verified the effective removal of cellular components, with the residual DNA content below the detection limit.

[0061] 2.2 Cell seeding and dynamic culture efficiency The dynamic culture system demonstrated excellent operational convenience. After inoculating the cell suspension, the decellularized scaffold could quickly integrate with the culture platform, and the total assembly time of the system was less than 5 minutes. Preliminary evaluation showed that the cell retention rate in the scaffold was approximately 75%, indicating good seeding efficiency. The process of changing the platform medium was similar to that of conventional plate culture, reducing the operation learning threshold. In 10 validation experiments, only 1 case failed due to medium contamination, and no common problems such as air bubbles infiltrating into the scaffold or medium leakage occurred.

[0062] 2.3 Morphological changes and cell distribution As Figure 6As shown in A, cells in the dynamic culture group showed characteristic dendritic distribution along the scaffold vascular network, and the tissue volume was estimated to be approximately 300 mm³ based on the size of the cell clusters. As the culture time increased, the cells gradually broke through the initial vascular network boundary and expanded. In contrast, the cell cluster morphology in the static culture group remained relatively constant throughout the culture period.

[0063] 2.4 Histology and cell behavior assessment Histological analysis provided strong evidence for the effectiveness of the platform. H&E staining showed that the cells in the dynamic culture group had intact morphology, and the dark staining of the nuclei indicated vigorous activity ( Figure 6 B). Clear luminal structures were observed in the dynamic culture samples to facilitate the perfusion of the culture medium, while such structures were absent in the static control group. In addition, nuclear lysis occurred in the static culture group, indicating impaired cell function and a significant tendency to apoptosis. Immunofluorescence analysis further revealed that Ki67 staining showed that the number of proliferating cells in the dynamic culture group was more than 5 times higher than that in the static group ( Figure 6 C, Figure 7 A); TUNEL assay showed that the apoptosis rate in the dynamic culture group was significantly reduced, and the apoptosis rate in the static group was as high as 32 times that of the dynamic group ( Figure 6 D, Figure 7 B).

[0064] 2.5 Engineering liver functional assessment To evaluate the functionality of tissue-engineered liver, we tested two key liver function indicators: albumin (ALB) synthesis and urea production. ALB immunofluorescence staining showed that the expression level of the dynamic culture group was significantly higher than that of the static group ( Figure 6 E). Quantitative fluorescence analysis showed that the expression of ALB in the dynamic group was about 1.5 times that of the static group ( Figure 7 C). Urea secretion further confirmed the liver function advantage of the dynamic group: the cumulative urea secretion of the dynamic group showed an S-shaped curve. Although the daily secretion of both groups showed a downward trend, the dynamic group always maintained a higher level ( Figure 8 These two indicators together verify the significant advantages of the dynamic culture system in supporting liver-specific functions.

[0065] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0066] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

Claims

1. A dynamic culture system for decellularized scaffold tissue, characterized in that: include: An outer shell (1) having an oxygen pipeline (3) disposed on its side wall; A culture dish (2) stacked on the outer shell (1), wherein two interfaces are provided on the bottom wall of the culture dish (2); A perfusion system is located in the housing (1), the perfusion system comprising an oxygenator and a peristaltic pump (4), the oxygenator comprising an aeration shell (5) and a polytetrafluoroethylene hollow fiber tube (6) located in the aeration shell (5), the surface of the polytetrafluoroethylene hollow fiber tube (6) having a pore structure, one end of the polytetrafluoroethylene hollow fiber tube (6) being connected to one of the interfaces, and the other end being connected to the peristaltic pump (4), the peristaltic pump (4) being further connected to another of the interfaces; the aeration shell (5) being in communication with the oxygen pipeline (3), and the oxygen pipeline (3) being in communication with an oxygen supply device; A connector, located in the culture dish (2), the connector being in communication with an interface connected to a peristaltic pump (4), the connector being used to connect the decellularized scaffold tissue placed in the culture dish (2); A power supply (7) electrically connected to the peristaltic pump (4) and used to provide power supply to the peristaltic pump (4); A controller for controlling the operation of the peristaltic pump (4).

2. The dynamic culture system of decellularized scaffold tissue according to claim 1, characterized in that: The oxygen supply device is an oxygen cylinder with a flow meter or an oxygen generator.

3. The dynamic culture system of decellularized scaffold tissue according to claim 1, characterized in that: The polytetrafluoroethylene hollow fiber tube (6) is spiral or wavy.

4. The dynamic culture system of decellularized scaffold tissue according to claim 1, characterized in that: The polytetrafluoroethylene hollow fiber tube (6) has an inner diameter of 0.9-1 mm, an outer diameter of 1.6-1.7 mm, a porosity of more than 60%, a pore size range of 0.2-50 μm, and a length of 1.8-2 m.

5. The dynamic culture system of decellularized scaffold tissue according to claim 1, characterized in that: The top of the aeration shell (5) is semi-closed.

6. A dynamic culture method for decellularized scaffold tissue, characterized in that: The method is carried out using the dynamic culture system according to any one of claims 1 to 4, wherein the culture method comprises: S1. Preparation of decellularized scaffold tissue; S2, pre-perfusing the decellularized scaffold tissue with a culture medium containing cells corresponding to the tissue; S3, inserting a silicone tube into the blood circulation inlet of the decellularized scaffold tissue, and then connecting it to the connector; S4, placing the dynamic culture system in a CO2 incubator and statically culturing for 4-5 hours to promote cell attachment; S5. Start the peristaltic pump (4) for continuous perfusion, supplement oxygen simultaneously, and replace the culture medium daily.

7. The dynamic culture method according to claim 5, characterized in that: The flow rate of the peristaltic pump (4) was set to 1.5-2 mL / min.

8. The dynamic culture method according to claim 5, characterized in that: The volume concentration of supplemented oxygen is 88%~90%, and the flow rate is 20~25 mL / min.

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