A cell scaffold for inducing adherent cells to aggregate to form three-dimensional cell spheroids, and a preparation method and application thereof

Abstract

The present application relates to the technical field of cell scaffold, in particular to a cell scaffold for inducing adherent cells to aggregate into three-dimensional cell spheroids, a preparation method and application thereof. The cell scaffold for inducing adherent cells to aggregate into three-dimensional cell spheroids provided by the present application takes the decellularized natural leaf vein as a skeleton and electrospun EVOH nanofiber as a functional layer, and cooperatively plays the natural topological guiding role of the leaf vein and the bioadhesion promoting role of the nanofiber through the micro-nano composite three-dimensional porous structure, so that a variety of adherent cells can be efficiently induced to autonomously aggregate into high-activity and uniform three-dimensional cell spheroids without adding exogenous growth factors; the preparation process is simple and mild, avoids the problem of pore blockage, and exhibits excellent induction spheroidization capacity and biocompatibility for adherent cells of different sources.

Description

Technical Field

[0001] This invention relates to the field of cell scaffold technology, specifically to a cell scaffold that induces adherent cells to aggregate and form three-dimensional cell spheres, its preparation method, and its application. Background Technology

[0002] Two-dimensional cell culture is currently the most common culture method in biomedical research. However, this culture mode causes cells to grow in a flattened form, lacking the three-dimensional spatial structure of real tissues in vivo. It also suffers from problems such as loss of cell polarity, simplistic proliferative behavior, and weak intercellular interactions, resulting in cell models that differ significantly from the actual physiological and pathological environment in humans. Adherent cells such as human choriocarcinoma cells JEG-3, human liver cancer cells HepG-2, human colon cancer cells Caco-2, and human embryonic lung fibroblasts MRC-5 are classic in vitro cell models, widely used in biomedical research, and there is a high demand for efficient and stable cell scaffolds.

[0003] However, current cell scaffolds used to induce cell spheroidization have certain limitations. Traditional synthetic cell scaffolds have simple structural designs, low biomimicry, and limited cell affinity, making it difficult to effectively guide adherent cells to aggregate autonomously. This results in low spheroidization rates, inconsistent spheroid morphology, and unstable bioactivity. Hydrogel scaffolds suffer from poor structural stability and are prone to collapse and deformation during long-term culture. Furthermore, most existing composite scaffolds involve cumbersome fabrication processes and stringent conditions, often employing impregnation methods, which can easily lead to pore blockage and uneven adhesion of functional layers. These problems further reduce the quality and reproducibility of cell scaffold construction, significantly limiting their practical application.

[0004] Therefore, developing a novel cell scaffold with excellent biocompatibility that can efficiently induce various adherent cells to spheroidize has become an urgent need to solve these problems. Summary of the Invention

[0005] Based on this, the purpose of this invention is to provide a cell scaffold that induces adherent cells to aggregate and form three-dimensional cell spheres, as well as its preparation method and application.

[0006] To achieve the above objectives, the present invention can adopt the following technical solutions: The present invention provides a cell scaffold for inducing adherent cells to aggregate and form three-dimensional cell spheres, comprising: a natural leaf vein skeleton treated with decellularization; and an ethylene-vinyl alcohol copolymer nanofiber layer composited on the surface of the natural leaf vein skeleton and inside its pores; the natural leaf vein skeleton and the ethylene-vinyl alcohol copolymer nanofiber layer constitute a three-dimensional porous composite structure.

[0007] Preferably, in the above-mentioned cell scaffold, the ethylene-vinyl alcohol (PVA-co-PE) copolymer nanofibers are deposited in situ on the natural leaf vein skeleton by electrospinning and are randomly and uniformly distributed along the mesh pores of the skeleton.

[0008] Another aspect of the present invention provides a method for preparing the above-mentioned cell scaffold, the method comprising: (1) decellularizing natural leaf veins to obtain a leaf vein skeleton with interconnected pores; (2) depositing ethylene-vinyl alcohol copolymer nanofibers on the surface of the leaf vein skeleton and inside the pores by electrospinning to form a composite scaffold; (3) drying the composite scaffold to obtain the cell scaffold.

[0009] Preferably, in the above preparation method, the decellularization process includes: selecting intact plant leaf veins and sequentially performing washing, degreasing, decellularization, impurity removal, and drying.

[0010] Preferably, in the above preparation method, the spinning solution for electrospinning is prepared by dissolving the PVA-co-PE (EVOH) polymer in a water / isopropanol mixed solvent.

[0011] More preferably, in the above preparation method, the concentration of the PVA-co-PE polymer is 5% to 10% w / v; and / or the volume ratio of the water / isopropanol mixed solvent is (1 to 3): (7 to 9).

[0012] Preferably, in the above preparation method, the electrospinning conditions include: a feed rate of 1 mL / h to 3 mL / h, a voltage of 20 kV to 30 kV, and a receiving distance of 10 cm to 20 cm.

[0013] In another aspect, the present invention provides the application of the above-mentioned cell scaffold or the cell scaffold prepared by the above-mentioned preparation method in the preparation of three-dimensional cell models, tissue regeneration and repair materials or drug screening models.

[0014] In another aspect, the present invention provides a three-dimensional cell model, which is obtained by seeding adherent cells onto the aforementioned cell scaffold.

[0015] Preferably, in the above three-dimensional cell model, the adherent cells are selected from at least one of human chorionic villus carcinoma JEG-3, human liver cancer cells HepG-2, human colon cancer cells Caco-2, and human embryonic lung fibroblasts MRC-5.

[0016] The beneficial effects of this invention include: The cell scaffold provided by this invention, which induces adherent cells to aggregate and form three-dimensional cell spheres, uses decellularized natural leaf veins as the framework and electrospun EVOH nanofibers as the functional layer. Through the synergistic effect of the micro-nano composite three-dimensional porous structure, it can efficiently induce various adherent cells to autonomously aggregate and form highly active and uniform three-dimensional cell spheres without the addition of exogenous growth factors. Its preparation process is simple and mild, avoiding the problem of pore blockage. It also shows excellent ability to induce sphere formation and biocompatibility for adherent cells from different sources such as JEG-3, HepG-2, Caco-2, and MRC-5. It provides a structurally stable and universally applicable scaffold solution for the construction of three-dimensional cell models and tissue engineering applications. Attached Figure Description

[0017] Figure 1 The surface morphology of the novel cell scaffold prepared in Example 1; Figure 2 The cell morphology of JEG-3 cells in Example 2 after 72 hours of culture on a cell scaffold; Figure 3 This is a bright-field image of JEG-3 cells in Comparative Example 1 cultured on decellularized natural leaf vein substrate for 48 hours. Figure 4 This is a bright-field image of JEG-3 cells cultured in EVOH nanofiber membrane for 48 hours in Comparative Example 2. Figure 5 This is a fluorescence image of live / dead staining of JEG-3 cells after 72 hours of culture on a cell scaffold in Example 2; Figure 6 This is a fluorescence image of HepG-2 cells after 72 hours of culture on a cell scaffold in Example 2, showing live / dead staining. Figure 7 This is a fluorescence image of live and dead cells stained after 72 hours of culture on a cell scaffold in Example 2. Figure 8 This is a fluorescence image of MRC-5 cells cultured on a cell scaffold for 72 hours in Example 2, showing live / dead staining. Figure 9 Comparison of cytotoxicity of the novel cell scaffold prepared in Example 1, the decellularized natural leaf vein substrate prepared in Comparative Example 1, and the EVOH nanofiber membrane prepared in Comparative Example 2. Figure 10 This is a quantitative statistical analysis of the spheroidization rate and cell spheroid diameter distribution of JEG-3 cells after 72 hours of culture on a cell scaffold in Example 2. Detailed Implementation

[0018] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0019] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0020] In a first aspect, embodiments of the present invention provide a cell scaffold for inducing adherent cells to aggregate and form three-dimensional cell spheres, comprising: a natural leaf vein skeleton treated with decellularization; and an ethylene-vinyl alcohol copolymer nanofiber layer composited on the surface of the natural leaf vein skeleton and inside its pores; the natural leaf vein skeleton and the ethylene-vinyl alcohol copolymer nanofiber layer constitute a three-dimensional porous composite structure.

[0021] It should be noted that existing two-dimensional culture dishes can only achieve a single layer of cells, lacking cell polarity and intercellular interactions. Traditional artificially synthesized single-spun membranes and hydrogel scaffolds have low biomimicry and are prone to pore blockage. Impregnation composite processes easily cause pore closure, resulting in low cell spheroidization rate, uneven spheroid size, and easy collapse during long-term culture. This invention uses decellularized natural leaf veins as a macroscopic support substrate. Electrospun EVOH nanofibers in situ permeate and coat all pores inside and outside the leaf veins, constructing a natural macroscopic network framework + multi-level interconnected three-dimensional porous composite structure of nano-biomimetic fibers. Natural leaf veins have their own original continuous multi-level interconnected grooves and pores, providing macroscopic transport channels for nutrient and metabolite exchange; EVOH nanofibers uniformly cover the pore walls, mimicking the nanotopology of the extracellular matrix, increasing the number of cell adhesion sites. This composite structure is non-cytotoxic and has excellent biocompatibility. It can break the two-dimensional flat growth pattern of adherent cells and guide them to migrate and aggregate autonomously inward. It can form uniform, highly active three-dimensional cell spheres in 72 hours. Fluorescent live-death staining in the examples confirmed that the apoptosis level of cells on the scaffold is extremely low, making it suitable for biomedical scenarios such as three-dimensional tumor models, epithelial barrier models, and drug toxicology screening.

[0022] In some specific examples, in the above-mentioned cell scaffold, the ethylene-vinyl alcohol copolymer nanofibers are deposited in situ on the natural leaf vein skeleton by electrospinning and are randomly and uniformly distributed along the mesh pores of the skeleton.

[0023] It should be noted that, unlike traditional soaking and coating composite methods, this invention uses an in-situ electrospinning process with a roller to fix the decellularized leaf veins to the receiving roller. The EVOH spinning jet can penetrate the surface of the leaf veins and reach deep into the pores of each level of groove. The fibers simultaneously coat the outer surface of the leaf veins and the inner wall of the pores, avoiding the problem of only surface coating and exposed and blocked internal pores. The nanofibers are evenly spread along the original network of the leaf veins, forming multi-level continuous channels of micron-sized macropores and nano-sized mesopores. Sufficient nano-adhesion sites provide a basis for the migration and aggregation of adherent cells, ensuring that cells form spheres synchronously and with uniform sphere size throughout the entire scaffold.

[0024] In a second aspect, embodiments of the present invention provide a method for preparing the above-mentioned cell scaffold, the method comprising: (1) decellularizing natural leaf veins to obtain a leaf vein skeleton with interconnected pores; (2) depositing ethylene-vinyl alcohol copolymer nanofibers on the surface of the leaf vein skeleton and inside the pores by electrospinning to form a composite scaffold; (3) drying the composite scaffold to obtain the cell scaffold.

[0025] It should be noted that the preparation method of this invention is mild and simple, requiring no high temperature, cross-linking agents, or complex post-treatment. The first step involves decellularization to remove endogenous plant proteins, lipids, and nucleic acids, eliminating plant-derived biological interference and completely preserving the original three-dimensional interconnected network of pores in the leaf veins. The second step involves in-situ permeation and composite bonding via roller electrospinning, achieving full-domain nanofiber modification of both internal and external pores. The third step involves room-temperature drying and curing, maintaining the complete three-dimensional structure of the leaf vein skeleton and preventing pore collapse. The process allows for standardized mass production, resulting in highly reproducible finished product structures and avoiding the defects of pore blockage and uneven fiber distribution caused by impregnation and composite bonding.

[0026] In some specific examples, the decellularization process in the above preparation method includes: selecting intact plant leaf veins and sequentially performing washing, degreasing, decellularization, impurity removal, and drying.

[0027] It should be noted that intact leaf veins ensure the original continuous network of grooves does not break; rinsing with clean water removes surface dust and epidermal impurities; the degreasing process removes hydrophobic lipids from the leaf veins, preventing the hydrophobic barrier from inhibiting cell adhesion; the decellularization step thoroughly degrades plant cell components, eliminating impurities and proteins that could trigger cell stress and apoptosis; impurity removal and filtration degrade residues prevent pore blockage; low-temperature drying and shaping maintain the three-dimensional interconnected porous framework of the leaf veins without shrinking or collapsing; the treated leaf vein substrate is non-cytotoxic and serves as a rigid support substrate suitable for subsequent electrospinning composites.

[0028] In some specific examples, the spinning solution used for electrospinning in the above preparation method is prepared by dissolving the PVA-co-PE polymer in a water / isopropanol mixed solvent.

[0029] It should be noted that pure water as a single solvent has a high viscosity, which can easily lead to the formation of large droplets and fiber beads during electrostatic stretching; pure isopropanol has poor solubility for PVA-co-PE, and polymer precipitation is easily formed. Using a water + isopropanol compound system allows for precise control of solution viscosity and surface tension. Complete polymer dissolution can be achieved by constant temperature stirring at 95℃ for 6 hours. The solution exhibits good stability, continuous electrostatic jetting without fiber breakage, and rapid solvent evaporation after deposition in the leaf vein pores, preventing long-term wetting and damage to the porous structure of the leaf veins, thus ensuring uniform nanofiber formation.

[0030] In some specific examples, in the above preparation method, the concentration of the PVA-co-PE polymer is 5% to 10% w / v; and / or the volume ratio of the water / isopropanol mixed solvent is (1 to 3): (7 to 9).

[0031] It should be noted that the polymer concentration is preferably 5%–10% w / v, such as 5% w / v, 7% w / v, or 10% w / v. Concentrations below 5% w / v result in too few polymer molecular chains, leading to brittle and easily broken spun fibers that cannot form a continuous adhesion network. Concentrations above 10% w / v result in excessively high solution viscosity, hindering jet stretching and causing coarse fibers and numerous beads to clog leaf vein pores. The water / isopropanol volume ratio can be selected as 1:9, 2:8, or 3:7. Too high a water ratio increases the solution surface tension, leading to jet instability; too high an isopropanol ratio reduces solubility and promotes precipitation. This formulation allows for the stable preparation of defect-free, uniform EVOH nanofibers.

[0032] In some specific examples, the electrospinning conditions in the above preparation method include: a feed rate of 1 mL / h to 3 mL / h, a voltage of 20 kV to 30 kV, and a receiving distance of 10 cm to 20 cm.

[0033] It should be noted that in the preparation method of this invention, the injection rate can be 1 mL / h to 3 mL / h, such as 1 mL / h, 2 mL / h, or 3 mL / h; the voltage can be 20 kV to 30 kV, such as 20 kV, 28 kV, or 30 kV; and the receiving distance can be 10 cm to 20 cm, such as 10 cm, 15 cm, or 20 cm. If the flow rate is too low, the mass production efficiency is low; if the flow rate is too high, the jet stretching is insufficient, resulting in coarse fibers; if the voltage is below 20 kV, the electrostatic stretching force is insufficient, resulting in larger fiber diameters; if it is above 30 kV, fly filaments and local fiber accumulation are likely to occur; if the receiving distance is too short, the solvent cannot evaporate in time, causing fiber adhesion and blockage of leaf vein pores; if the distance is too long, the fiber deposition is insufficient, failing to completely cover the pore walls. This parameter window allows the nanofibers to penetrate into all grooves of the leaf veins and be uniformly distributed throughout the entire area.

[0034] Thirdly, embodiments of the present invention provide an application of the cell scaffold described above or prepared by the above-described method in the preparation of three-dimensional cell models, tissue regeneration and repair materials, or drug screening models.

[0035] It should be noted that the cell scaffold of this invention can be used for the construction of three-dimensional cell models. Specifically, it can induce adherent cells such as JEG-3, HepG-2, Caco-2, or MRC-5 to form uniformly sized, highly active three-dimensional cell spheres after 72 hours, compensating for the deficiencies of two-dimensional plate cells, such as lack of cell polarity and weak intercellular interactions. In addition, the cell scaffold of this invention can also be used as a tissue regeneration and repair material. The EVOH nanofiber biomimetic extracellular matrix, with its large pores facilitating cell ingrowth, can be used for the in vitro preconstruction of epithelial and mesenchymal tissues. Furthermore, the cell scaffold of this invention can also be used for drug toxicology / anti-tumor screening models. The three-dimensional tumor spheres better match the proliferation and microenvironment characteristics of solid tumors in vivo, resulting in more reliable drug response data and significantly improving the correlation between in vitro screening and in vivo experiments.

[0036] Fourthly, embodiments of the present invention provide a three-dimensional cell model, which is obtained by seeding adherent cells onto the aforementioned cell scaffold.

[0037] It should be noted that conventional culture dishes can only achieve a single layer of cells, making it difficult to form dense cell spheres. This composite scaffold combines the macropores of leaf veins with an EVOH nanoadhesion network. After cell seeding, cells can adhere along the nanofibers and migrate and aggregate into the pores. After 72 hours of culture, well-structured three-dimensional cell spheres can be formed. Live and dead cell staining shows that the proportion of green fluorescence is extremely high, while the red fluorescence of apoptosis is very low, indicating excellent cell viability. The constructed three-dimensional model highly simulates the intercellular communication, proliferation and metabolic characteristics of tissues in vivo, making it suitable for in vitro basic research on tumor mechanisms, epithelial barriers, fibrosis, and other related fields.

[0038] In some specific examples, in the above three-dimensional cell model, the adherent cells are selected from at least one of human chorionic villus carcinoma JEG-3, human liver cancer cells HepG-2, human colon cancer cells Caco-2, and human embryonic lung fibroblasts MRC-5.

[0039] It should be noted that the above four types are all classic adherent cells in the biomedical field, which are difficult to aggregate into spheres autonomously in traditional two-dimensional culture; when the above cells are cultured for 72 hours using the cell scaffold of this invention, they all form uniform and highly active cell spheres: JEG-3 is used for placental physiology / tumor three-dimensional models; HepG-2 is used to construct liver metabolism and drug toxicity models; Caco-2 simulates the intestinal epithelial barrier; MRC-5 is used for pulmonary fibrosis and interstitial tissue research, covering the mainstream in vitro modeling needs of tumors, epithelium, and mesenchyme, with a wide range of applicable cells and good model reproducibility.

[0040] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0041] Product Preparation The following example illustrates the preparation method of decellularized natural leaf vein substrate: Fresh poplar leaves were boiled in a 5% sodium hydroxide (Aladdin, China) solution for 30 minutes until they turned yellowish-brown. The boiled leaves were then removed and rinsed with clean water. The mesophyll tissue was gently brushed with a soft toothbrush until a complex and clear vein network appeared. Finally, the vein tissue was bleached in a 2% hydrogen peroxide solution for 24 hours and dried to obtain a porous, decellularized leaf vein substrate.

[0042] Example 1 (1) PVA-co-PE polymer (ethylene-vinyl alcohol copolymer precursor, 414093-100G, Merck, China) was dissolved in a water / isopropanol mixed solution at a concentration of 7% (w / v), wherein the volume ratio of the water / isopropanol mixed solution was 2:8; the above mixture was placed in an oil bath at 95°C, and a magnetic rotor was added to seal and dissolve for 6 hours until the polymer was completely dissolved, to obtain a uniform and transparent EVOH nanofiber spinning solution.

[0043] (2) The obtained EVOH nanofiber spinning solution was loaded into a 5mL syringe with a metal needle (0.8mm inner diameter) and the syringe was installed on the electrospinning equipment. The injection rate was controlled to be 2mL / h by the injection pump. The decellularized natural leaf vein substrate was flattened and fixed on the surface of the stainless steel roller receiving device, and the distance between the needle and the roller was adjusted to 15cm. The high voltage power supply was turned on and a voltage of 28kV was applied to form a high voltage electric field between the needle and the roller. The charged polymer jet was ejected from the needle and stretched and refined under the action of the electric field to form nanofibers, which were uniformly deposited on the surface of the natural leaf vein substrate and inside the three-dimensional interconnected pores. The electrospinning time was controlled to be 10 minutes so that the amount of EVOH nanofibers deposited was about the volume corresponding to 1mL of spinning solution.

[0044] (3) The composite scaffold deposited with EVOH nanofibers is placed at room temperature to dry and solidify naturally, thus obtaining a cell scaffold that induces adherent cells to aggregate and form three-dimensional cell spheres.

[0045] Example 2 (1) Human chorionic villus carcinoma JEG-3, human hepatocellular carcinoma HepG-2, human colon cancer Caco-2 and human embryonic lung fibroblast MRC-5 were taken respectively (all of which could be purchased from the American Type Culture Collection Center (ATCC) or the Chinese Academy of Sciences Cell Bank).

[0046] (2) Digest the above cells with a confluence of more than 80% in the T25 cell culture flask. Add 1 mL of 0.25% trypsin-EDTA digestion solution and digest at 37°C for 2-5 minutes. After the cells detach from the cell wall, add DMEM / F12 complete culture medium containing 10% fetal bovine serum to stop the digestion. Transfer the cell suspension to a centrifuge tube, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and resuspend the cell pellet in fresh complete culture medium.

[0047] (3) Take 10 μL of cell suspension and drop it onto a cell counting slide, then count the cells using a cell counter; based on the counting results, dilute the cells to a suitable concentration (approximately 1 × 10⁻⁶) with complete culture medium. 5 (cells / mL).

[0048] (4) Cut the novel cell scaffold prepared in Example 1 to a suitable size (about 1cm×1cm) and place it at the bottom of a 24-well cell culture plate; add 1mL of diluted cell suspension to each well so that the cells are evenly seeded on the surface of the scaffold; place the 24-well plate in a CO2 incubator and culture it at 37°C, 5% CO2, and saturated humidity for 72 hours to allow the cells to aggregate and grow, and obtain three-dimensional cell spheres of different cell types.

[0049] Comparative Example 1 The same batch of decellularized natural leaf vein substrate as in Example 1 was selected and used directly for subsequent cell culture tests without electrospinning EVOH nanofiber deposition treatment.

[0050] Comparative Example 2 Using the same electrospinning process conditions as in Example 1, EVOH nanofibers were directly collected on a stainless steel collector to form an EVOH nanofiber membrane that did not conform to the natural leaf vein skeleton. This membrane served as a control scaffold for subsequent cell culture testing.

[0051] Characterization test The surface morphology of the novel cell scaffold for inducing cell spheroidization prepared in Example 1 is shown in the figure below. Figure 1 As shown, the results indicate that EVOH nanofibers uniformly cover the surface of the natural leaf vein skeleton, and the fibers are distributed in a three-dimensional network. The porous network structure of the natural leaf veins is completely preserved, and the nanofibers can penetrate deep into the pores of the leaf veins to form a composite scaffold with a multi-level three-dimensional structure.

[0052] The cell morphology diagram of the three-dimensional cell spheroids of JEG-3 cells prepared in Example 2 is shown below. Figure 2 As shown, the results indicate that JEG-3 cells are evenly distributed on the cell scaffold, and the cells exist in a distinct three-dimensional spherical shape with complete spherical structure and clear boundaries. This demonstrates that the novel cell scaffold provided by this invention can effectively induce JEG-3 adherent cells to autonomously aggregate and form uniform three-dimensional spherical structures.

[0053] Application testing (a) Staining of live and dead cells (1) After culturing different cells in Example 2 above for 72 hours, discard the culture medium in the wells and gently wash the scaffold twice with phosphate-buffered saline (PBS); prepare the live and dead cell staining working solution: take Calcein-AM (calcein-AM, live cell green fluorescent staining agent, C2015S, Beyotime, China) and PI (propidium iodide, dead cell red fluorescent staining agent, C2015S, Beyotime, China), and dilute with PBS to the appropriate concentration according to the kit instructions.

[0054] (2) Add the staining working solution to the well and incubate at room temperature in the dark for 30 minutes. After incubation, remove the staining solution and wash twice with PBS. Place the scaffold under a fluorescence microscope to observe and take pictures. Live cells show green fluorescence and dead cells show red fluorescence.

[0055] Three-dimensional fluorescent images of live and dead cell spheroids of JEG-3 cells prepared in Example 2 are shown below. Figure 3 As shown, the results indicate that almost no red fluorescence signal was observed in the field of view, suggesting that JEG-3 cells did not experience significant cell death on the novel cell scaffold of this invention; the green fluorescence was significant and widely distributed, indicating that the cells within the cell spheroids maintained good biological activity. These results demonstrate that the novel cell scaffold of this invention has good biocompatibility and cell viability maintenance capabilities for JEG-3 cells.

[0056] Three-dimensional fluorescent images of live and dead cell spheroids of HepG-2 cells prepared in Example 2 are shown below. Figure 4 As shown, the results indicate that almost no red fluorescence signal was observed in the field of view, suggesting that HepG-2 cells did not experience significant cell death on the novel cell scaffold of this invention; the green fluorescence was significant and widely distributed, indicating that the cells within the cell spheroids maintained good biological activity. These results demonstrate that the novel cell scaffold of this invention has good biocompatibility and cell viability maintenance capabilities for HepG-2 cells.

[0057] Three-dimensional fluorescent images of live and dead cell spheroids of Caco-2 cells prepared in Example 2 are shown below. Figure 5 As shown, the results indicate that almost no red fluorescence signal was observed in the field of view, suggesting that Caco-2 cells did not experience significant cell death on the novel cell scaffold of this invention; the green fluorescence was significant and widely distributed, indicating that the cells within the cell spheroids maintained good biological activity. These results demonstrate that the novel cell scaffold of this invention has good biocompatibility and cell viability maintenance capabilities for Caco-2 cells.

[0058] Three-dimensional fluorescent images of live and dead cell spheroids of MRC-5 cells prepared in Example 2 are shown below. Figure 6 As shown, the results indicate that almost no red fluorescence signal was observed in the field of view, suggesting that MRC-5 cells did not experience significant cell death on the novel cell scaffold of this invention; the green fluorescence was significant and widely distributed, indicating that the cells within the cell spheroids maintained good biological activity. These results demonstrate that the novel cell scaffold of this invention has good biocompatibility and cell viability maintenance capabilities for MRC-5 cells.

[0059] Comparative Example 1 used the same batch of decellularized natural leaf vein substrate as Example 1, without electrospinning EVOH nanofiber deposition treatment. JEG-3 cells were added and cultured in a cell culture incubator for 48 hours. Figure 3 As shown, JEG-3 cells cannot attach to and grow on decellularized natural leaf vein substrate.

[0060] Comparative Example 2 used the exact same electrospinning process conditions as Example 1, directly collecting EVOH nanofibers on a stainless steel receiver to form an EVOH nanofiber membrane. This membrane did not incorporate the natural leaf vein framework. JEG-3 cells were then added and cultured in a cell culture incubator for 48 hours. Figure 4 As shown, JEG-3 cells also cannot attach to the EVOH nanofiber membrane for growth.

[0061] The combined results of the above cell culture experiments demonstrate that the novel cell scaffold provided by this invention can not only efficiently induce various adherent cells (including human choriocarcinoma cells JEG-3, human hepatocellular carcinoma cells HepG-2, human colon cancer cells Caco-2, and human embryonic lung fibroblasts MRC-5) to autonomously aggregate into three-dimensional cell spheroids, but also provide a favorable microenvironment for the cell spheroids, maintaining high cell viability. Furthermore, this novel cell scaffold exhibits good applicability to adherent cells from different sources and tissue types, demonstrating broad substrate compatibility and application potential.

[0062] (ii) Other verification tests The novel cell scaffold prepared in Example 1, which induces adherent cell aggregation to form three-dimensional cell spheres, the decellularized natural leaf vein substrate prepared in Comparative Example 1, and the EVOH nanofiber membrane prepared in Comparative Example 2 exhibit cytotoxicity similar to those described above. Figure 9 As shown, cell viability in all groups was higher than baseline, with the leaf group exhibiting significantly higher cell viability than the other groups (p<0.01). The inhibition rate was below 0 for all groups, indicating no significant cell proliferation inhibition effect. The leaf group showed the lowest inhibition rate, significantly different from other groups (p<0.01). These results confirm that the novel cell scaffold possesses excellent cell compatibility and can effectively promote cell survival.

[0063] In Example 2, the quantitative statistics of the spheroidization rate and the statistical statistics of the cell spheroid diameter distribution of JEG-3 cells after 72 hours of culture on a novel cell scaffold are as follows: Figure 10 As shown, the cell spheroid density on the cell scaffold can reach 1164 cells / mm². 2 Numerous cell spheroids can form on the scaffold surface. The diameter of the cell spheroids exhibits an approximately normal distribution, with an average diameter of 32.45 ± 11.06 μm. Most cell spheroids are concentrated in the 20–40 μm range, with micro-cell spheroids smaller than 10 μm and large cell spheroids larger than 45 μm accounting for a smaller proportion. The large standard deviation indicates significant variation in cell spheroid size on the scaffold surface. This size variation may be related to uneven cell seeding and the different local microenvironments provided by the porous leaf vein scaffold.

[0064] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A cell scaffold that induces adherent cells to aggregate and form three-dimensional cell spheres, characterized in that, include: Natural leaf vein framework after decellularization treatment; as well as An ethylene-vinyl alcohol copolymer nanofiber layer composited on the surface of the natural leaf vein skeleton and inside its pores; The natural leaf vein skeleton and the ethylene-vinyl alcohol copolymer nanofiber layer constitute a three-dimensional porous composite structure.

2. The cell scaffold according to claim 1, characterized in that, The ethylene-vinyl alcohol (PVA-co-PE) copolymer nanofibers are deposited in situ on the natural leaf vein skeleton by electrospinning and are randomly and uniformly distributed along the mesh pores of the skeleton.

3. The method for preparing the cell scaffold according to claim 1 or 2, characterized in that, Preparation methods include: (1) Natural leaf veins are decellularized to obtain a leaf vein skeleton with interconnected pores; (2) Ethylene-vinyl alcohol copolymer nanofibers are deposited on the surface of the leaf vein skeleton and inside the pores by electrospinning process to form a composite scaffold; (3) The composite scaffold is dried to obtain the cell scaffold.

4. The preparation method according to claim 3, characterized in that, The decellularization process includes: selecting intact plant leaf veins and sequentially performing washing, degreasing, decellularization, impurity removal, and drying.

5. The preparation method according to claim 3 or 4, characterized in that, The spinning solution for electrospinning is prepared by dissolving the PVA-co-PE polymer in a water / isopropanol mixed solvent.

6. The preparation method according to claim 5, characterized in that, The concentration of the PVA-co-PE polymer is 5%–10% w / v; and / or The volume ratio of the water / isopropanol mixed solvent is (1-3):(7-9).

7. The preparation method according to claim 3, 4 or 6, characterized in that, The conditions for electrospinning include: sample feed rate of 1 mL / h to 3 mL / h, voltage of 20 kV to 30 kV, and receiving distance of 10 cm to 20 cm.

8. The application of the cell scaffold according to claim 1 or 2 or the cell scaffold prepared by the preparation method according to any one of claims 3 to 7 in the preparation of three-dimensional cell models, tissue regeneration and repair materials or drug screening models.

9. A three-dimensional cell model, characterized in that, Adherent cells are obtained by seeding them onto the cell scaffold as described in claim 1 or 2.

10. The three-dimensional cell model according to claim 9, characterized in that, The adherent cells are selected from at least one of human chorionic villus carcinoma JEG-3, human liver cancer cells HepG-2, human colon cancer cells Caco-2, and human embryonic lung fibroblasts MRC-5.

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