Cell capsule bio-ink as well as preparation method and application thereof

Abstract

The invention relates to cell capsule bio-ink as well as a preparation method and application thereof. The preparation method comprises the following steps: step 1, preparing acrylic acid esterification modified cell membrane vesicles (mCMVs) from cell membrane vesicles and acrylate-polyethylene glycol-succinimido ester (AC-PEG-NHS); step 2, preparing active factor-loaded cell capsules (PUN (at) mCMVs) by using the acrylated and modified cell membrane vesicles (mCMVs) obtained in the step 1 and active factors; and step 3, mixing a methacrylic acid polymer material, a photoinitiator, living cells and the active factor-loaded cell capsules (PUN (at) mCMVs) obtained in the step 2 to form the photo-crosslinkable cell capsule bio-ink. The drug-loaded slow-release bio-ink provided by the invention not only is suitable for 3D bio-printing and can be arbitrarily formed, but also can slowly release loaded active factors into loaded cells in a targeted manner to a certain extent.

Description

Technical Field

[0001] This invention relates to the technical field of biomedical materials, and in particular to a cell capsule bio-ink, its preparation method and application, the application of which involves tissue engineering and 3D bioprinting. Background Technology

[0002] Tissue engineering, an interdisciplinary field combining materials science and life sciences, is dedicated to developing bioactive alternative materials. Through the construction of cell-material complexes, it aims to achieve functional repair and reconstruction of human tissues. This field integrates engineering and biomedical principles to provide innovative solutions for tissue regeneration. Among these, bio-inks used in 3D bioprinting, because they carry living cells, can better construct large-volume, irregular, and complex tissue structures, and together with 3D bioprinting, have wide applications in tissue engineering.

[0003] Bio-inks are defined as biomaterials containing bioactive components such as cells, growth factors, and active agents. Their physical and chemical properties must be compatible with 3D bioprinting processes to ensure molding accuracy. Material rheological properties, cell compatibility, and bioactivity are key parameters in bio-ink design. Current bio-inks suffer from limitations such as single functionality and insufficient adaptability, making it difficult to simultaneously meet the complex needs of 3D bioprinting and tissue engineering. Active agents are one of the four essential elements of tissue engineering; precise and targeted release is crucial. Developing a functional bio-ink capable of precisely and targetedly releasing active agents can overcome existing technological bottlenecks.

[0004] In recent years, the loading of bioactive factors on tissue engineering scaffold materials has attracted much attention, and hydrogels obtained through 3D bioprinting are also a type of scaffold material carrying living cells. However, lipid-soluble bioactive factors are difficult to mix with hydrophilic hydrogel scaffold materials due to their physical properties, while factors such as protein growth factors and functional RNA are easily degraded if simply physically mixed with hydrogel scaffolds, severely affecting their efficacy. Moreover, these delivery methods lack targeting and do not have a sustained-release effect. Summary of the Invention

[0005] This invention designs a cell capsule bio-ink and its preparation method and application. The technical problems it solves are: (1) Lipid-soluble active factors are difficult to mix with hydrophilic hydrogel scaffold materials due to their physical properties. If factors such as protein growth factors and functional RNA are simply physically mixed with hydrogel scaffolds, they are easily degraded, which seriously affects their efficacy. (2) The delivery methods of lipid-soluble active factors, protein growth factors, functional RNA and other factors lack targeting and do not have a sustained-release effect.

[0006] To solve the aforementioned technical problems, the present invention adopts the following solution: A method for preparing a cell capsule bio-ink includes the following steps: Step 1: Prepare acrylate-modified cell membrane vesicles (mCMVs) using cell membrane vesicles and acrylate-polyethylene glycol-succinimide (AC-PEG-NHS). Step 2: Using the acrylate-modified cell membrane vesicles (mCMVs) obtained in Step 1 and active factors, cell capsules loaded with active factors (PUN@mCMVs) are prepared. Step 3: Mix the methacrylated polymer material, photoinitiator, live cells, and cell capsules (PUN@mCMVs) loaded with active factors obtained in Step 2 to form a photocrosslinkable cell capsule bio-ink.

[0007] Preferably, in step 1, cell membrane vesicles are mixed with acrylate-polyethylene glycol-succinimide ester (AC-PEG-NHS) and acrylate-modified cell membrane vesicles (mCMVs) are prepared by a light-shielded reaction.

[0008] Preferably, the cell membrane vesicles in step 1 are derived from mammalian cell membranes, and more preferably, are cell membrane vesicles of the same type as the cells they carry.

[0009] Preferably, the reaction conditions in step 1 include: step 1 is carried out in PBS containing the photoinitiator LAP.

[0010] Preferably, in step 2, the acrylate-modified cell membrane vesicles (mCMVs) obtained in step 1 are mixed with active factors, and then successively dispersed by ultrasonication and extruded by a liposome extruder. After purification by ultracentrifugation, cell capsules loaded with active factors (PUN@mCMVs) are finally obtained.

[0011] Preferably, the liposome extruder in step 2 uses a porous membrane with decreasing pore size for stepwise extrusion.

[0012] Preferably, the active factor in step 2 is at least one of a lipid-soluble active factor, a growth factor, a drug, or a functional RNA, preferably punicin (PUN).

[0013] Preferably, the methacrylated polymer material in step 3 is at least one of methacrylated sericin (SerMA) and methacrylated gelatin.

[0014] Preferably, the living cells in step 3 are chondrocytes.

[0015] A cell capsule bio-ink, characterized in that it comprises a cell capsule (PUN@mCMVs) carrying active factors, a methacrylated polymer material, a photoinitiator, and live cells, wherein the cell capsule is internally loaded with active factors and has photocrosslinking properties.

[0016] Preferably, the lipid bilayer membrane of the cell capsule isolates the internal active factors from the external hydrogel environment, thus preventing the degradation of the active factors.

[0017] An application of a cell capsule bio-ink, characterized in that it is used for 3D bioprinting, forming a hydrogel scaffold through photopolymerization to achieve targeted sustained release of active factors.

[0018] Preferably, the 3D bioprinting is digital light processing (DLP) printing, and the printing parameters include a light intensity of 10-50 mW / cm². 2 The exposure time is 5-30 seconds.

[0019] Preferably, the hydrogel scaffold gradually releases active factors through enzymatic degradation during cell growth, and the cell capsule is homologous to the cell membrane of the loaded cells, thus achieving targeted delivery.

[0020] An application of a cell capsule bio-ink, characterized in that: it is used for 3D bioprinting, forming a hydrogel scaffold through photopolymerization, and the hydrogel scaffold is implanted in the body to become an artificial tissue or artificial organ.

[0021] The cell capsule bio-ink, its preparation method, and its application have the following beneficial effects: (1) This invention creates a drug-loaded sustained-release bio-ink that is not only suitable for 3D bioprinting and can be shaped arbitrarily, but also can target and release the active factors loaded onto the cells to a certain extent.

[0022] (2) In this invention, the lipid-soluble active factors / growth factors loaded in the cell capsules are encapsulated within cell membrane vesicles, which has a higher binding force compared to drug delivery methods distributed in the pores of hydrogels, thus avoiding burst release. (3) In this invention, the cell membrane surface retains natural receptor proteins, thus the cell capsule has a certain precise release and targeting function.

[0023] (4) In this invention, through multi-step processing of cell membrane vesicles, and by reacting with acrylate-polyethylene glycol-succinimide, cell membrane vesicles grafted with acrylic acid are obtained, which have the characteristics of copolymerization with methacrylic polymer materials.

[0024] (5) The present invention esterifies cell membrane vesicles with acrylic acid to give them photocrosslinking properties and constructs a cell capsule bio-ink system.

[0025] (6) The cell capsule bio-ink of the present invention can target and release lipid-soluble active factors / drugs, or encapsulate hydrophilic active factors / drugs / growth factors to reduce their interaction with the cells carried during the 3D bioprinting process.

[0026] (7) The cell capsule bio-ink of the present invention has pioneered the application of tissue repair in pathological microenvironments. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the synthetic route of cell capsule bio-ink carrying the active factor punicin in this invention.

[0028] Figure 2 This is the 1H NMR spectrum of the cell capsules (mCMVs) without active factors / growth factors in this invention.

[0029] Figure 3 This is a dynamic light scattering particle size distribution of cell capsules (PUN@mCMVs) carrying the active factor punicin in this invention.

[0030] Figure 4 Negative staining TEM image of cell capsules (PUN@mCMVs) loaded with the active factor punicin.

[0031] Figure 5 Image showing the antioxidant capacity of cells in different hydrogels.

[0032] Figure 6 3D bioprinting image of cell capsule bio-ink (PUN8@SerMA-mCMVs).

[0033] The following are the English abbreviations and their corresponding Chinese meanings in the examples: PUN: Puniculin (a fat-soluble active factor); LAP: Lithium phenyl-2,4,6-trimethylbenzoylphosphonate (photoinitiator); AC-PEG-NHS: Acrylate-polyethylene glycol-succinimide ester (chemical modification reagent). mCMVs: Modified cell membrane vesicles (acrylated vesicles); SerMA: Methacrylated sericin (a photocrosslinkable natural polymer material); PBS: Phosphate Buffer (a commonly used biological experimental buffer); TEM: Transmission electron microscope (used to observe nanoscale structures); DLP: Digital Light Processing (a photopolymer 3D printing technology); ROS: Reactive oxygen species (molecules associated with cellular oxidative stress). CMVs: Cell membrane vesicles (vesicle structures derived from the natural cell membrane). Detailed Implementation

[0034] The following is combined with Figures 1 to 6 The present invention will be further described as follows: like Figure 1 As shown, this invention uses chondrocytes as an example to illustrate the preparation of cell membrane vesicles, and uses pungent glycosides as an example to illustrate how acrylate-modified cell membrane vesicles can load lipid-soluble drugs / active factors to prepare cell capsules. Drug-loaded (lipophilic active factors, growth factors, drugs, or functional RNA) chondrocyte membrane vesicle-based cell capsules were prepared through steps such as ultrasonic dispersion, ultracentrifugation, acrylate-polyethylene glycol-succinimide modification, and ultrasonic drug loading. These pungent glycoside-coated cell capsules, together with methacrylated natural polymer materials, such as methacrylated sericin, and chondrocytes, constituted the cell capsule bio-ink.

[0035] Example 1: A method for preparing a cell capsule bio-ink, comprising the following steps: Step 1: Preparing acrylate-modified cell membrane vesicles (mCMVs) using cell membrane vesicles and acrylate-polyethylene glycol-succinimide ester (AC-PEG-NHS); Step 2: Preparing cell capsules loaded with active factors (PUN@mCMVs) using the acrylate-modified cell membrane vesicles (mCMVs) obtained in Step 1; Step 3: Mixing methacrylated polymer material, photoinitiator, live cells, and the cell capsules loaded with active factors (PUN@mCMVs) obtained in Step 2 to form a photocrosslinkable cell capsule bio-ink.

[0036] In step 1, cell membrane vesicles are mixed with acrylate-polyethylene glycol-succinimide (AC-PEG-NHS) at a mass ratio of 1:1 to 1:5 and reacted in the dark for 12-24 hours to prepare acrylate-modified cell membrane vesicles (mCMVs). The cell membrane vesicles in step 1 are derived from mammalian cell membranes, preferably chondrocyte membrane vesicles. The reaction conditions in step 1 include: the reaction is carried out in PBS containing 0.5 wt% photoinitiator LAP at a reaction temperature of 4°C.

[0037] Step 2: The acrylate-modified cell membrane vesicles (mCMVs) obtained in Step 1 are mixed with active factors at a mass ratio of 1:1 to 2:1. The mixture is then sequentially dispersed by ultrasonication and extruded using a liposome extruder. After centrifugation purification, cell capsules loaded with active factors (PUN@mCMVs) are finally obtained. The liposome extruder used in Step 2 employs porous membranes with pore sizes of 800 nm, 400 nm, 200 nm, and 100 nm for sequential extrusion.

[0038] By progressively reducing the pore size (800 nm → 400 nm → 200 nm → 100 nm), the size of cell membrane vesicles (such as mCMVs) and their loaded active factors (such as punicalin) is gradually reduced to the target range (approximately 100 nm). This process avoids the membrane clogging or uneven particle breakage problems that may occur when using the smallest pore size directly, ensuring a narrower and more uniform particle size distribution in the end.

[0039] Stepwise extrusion facilitates the binding of active factors to cell membrane vesicles through mechanical force. The shear force applied by the smaller pore size helps to more efficiently encapsulate lipid-soluble factors (such as punicalin) or hydrophilic factors (such as proteins) into the vesicles, while simultaneously adjusting the vesicle membrane structure to enhance loading stability. The uniform particle size (approximately 100 nm) is beneficial for subsequent uniform mixing with methacrylated polymers (such as SerMA), ensuring the structural uniformity of the photocrosslinked hydrogel. Furthermore, the uniformly sized cell capsules exhibit better flowability during 3D bioprinting, reducing the risk of printhead clogging and improving printing accuracy.

[0040] The active factor mentioned in step 2 is at least one of a lipid-soluble active factor, a growth factor, a drug, or a functional RNA, preferably punicin (PUN).

[0041] The methacrylated polymer material mentioned in step 3 is at least one of methacrylated sericin (SerMA) and methacrylated gelatin. The living cells mentioned in step 3 are chondrocytes. Example 2:

[0042] Step 1: Dissolve an appropriate amount of chondrocyte membrane vesicles in PBS containing 0.5 wt% LAP, add an appropriate amount of AC-PEG-NHS, with a mass ratio of vesicles to AC-PEG-NHS of 1:2, and vortex in the dark for 5 minutes to dissolve.

[0043] The reaction mixture was filtered using a 0.22 μm filter and then incubated at 4 °C in the dark for 24 hours to obtain modified chondrocyte membrane vesicles mCMVs.

[0044] Step 2: Modified chondrocyte membrane vesicles (mCMVs) and the lipid-soluble active factor punicalin (PUN) were mixed in PBS at a mass ratio of 1:1-2:1, and then dispersed in ice water by sonication. The mixture was then repeatedly extruded through porous membranes at 800 nm, 400 nm, 200 nm, and 100 nm using a liposome extruder. Cell capsules loaded with punicalin were collected by centrifugation at 100,000 g for 30-60 minutes at 4 °C. The ice water could be a mixture of ice and water at 0 °C, preferably deionized water or double-distilled water.

[0045] Step 3: Mix 1.5 g of photopolymerizable polymer material SerMA, cell capsules of different masses PUN@mCMVs, and a certain number of chondrocytes in 10 mL of PBS containing LAP (0.5 wt%, pH 7.4) or cell culture medium containing LAP (0.5% wt%, pH 7.4) to prepare cell capsule bio-ink solution.

[0046] In step 3, the methacrylated polymer material has a mass percentage content of 10%-20%, the photoinitiator has a mass percentage content of 0.5%, the live cell content is 10 million / mL, and the cell capsules loaded with active factors obtained in step 2 have a mass percentage content of 0.1% to 0.5%, with the remainder being PBS solution or cell culture medium. Example 3:

[0047] The cell capsule bio-ink prepared by this invention comprises cell capsules carrying active factors (PUN@mCMVs), methacrylated polymer materials, photoinitiators, and live cells. The cell capsules encapsulate active factors and have photocrosslinking properties. The lipid bilayer membrane of the cell capsules isolates the internal active factors from the external hydrogel environment, preventing the degradation of the active factors. Example 4:

[0048] The first application of the cell capsule bio-ink of this invention is for 3D bioprinting, which forms a hydrogel scaffold through photopolymerization to achieve targeted sustained release of active factors.

[0049] 3D bioprinting uses digital light processing (DLP) printing, with printing parameters including light intensity of 10-50 mW / cm². 2 The exposure time is 5-30 seconds. During cell growth, the hydrogel scaffold gradually releases active factors through enzymatic degradation, and the cell capsules are homologous to the cell membranes of the loaded cells, achieving targeted delivery. Example 5:

[0050] The second application of the cell capsule bio-ink of this invention is for 3D bioprinting, where it forms a hydrogel scaffold through photopolymerization, and the hydrogel scaffold is implanted in the body to become an artificial tissue or artificial organ.

[0051] like Figures 2 to 6 As shown, physicochemical test results indicate that the cell capsule bio-ink hydrogel possesses excellent rheological and mechanical properties, swelling rate, and degradation rate. This bio-ink exhibits good in vitro biocompatibility and no in vivo immunogenicity. By optimizing relevant printing parameters, the printing of complex patterns was successfully achieved.

[0052] Figure 2The proton NMR spectroscopy directly confirmed that: acrylate groups were successfully grafted (the appearance of the double bond peak at δ 5.8-6.4 ppm); the PEG segment was retained (the presence of the methylene peak at δ 3.6-3.8 ppm); and the membrane structure remained intact (the natural lipid signal was not lost).

[0053] Figure 2 The NMR spectrum of unloaded mCMVs showed characteristic lipid peaks: chemical shifts of long-chain alkanes (CH2) (δ 1.2–1.5 ppm); characteristic peaks of phospholipid head groups (such as phosphocholine) (δ 3.0–3.5 ppm); and hydroxyl peaks of cholesterol (δ 0.5–1.0 ppm). These signals collectively indicate that the main components of the vesicle membrane are lipids (phospholipids, cholesterol, etc.), suggesting the presence of a bilayer membrane structure.

[0054] like Figure 3 The image shows the dynamic light scattering (DLS) particle size distribution of cell capsules (PUN@mCMVs) loaded with pungent glycosides.

[0055] The particle size distribution shows a peak concentration of 100-200 nm, indicating good size uniformity of the cell capsules, meeting the requirements of nanoscale drug delivery systems. Uniform particle size ensures the rheological properties of the bio-ink (such as shear thinning), preventing printhead clogging. Nanoscale particles provide a large specific surface area, facilitating the gradual release of active ingredients. The narrow distribution indicates that vesicles have not aggregated or ruptured, supporting the integrity of the lipid bilayer structure.

[0056] Polydispersity Index (PDI): A PDI value < 0.3 indicates a highly uniform particle distribution, confirming the effectiveness of the preparation process (such as step-by-step filtration in a liposome extruder).

[0057] like Figure 4 As shown, the morphological characteristics are: clearly defined circular or near-circular structures with distinct boundaries; a typical bilayer membrane structure (parallel dark lines enclosing bright areas) is visible at the edges, directly proving the existence of the lipid bilayer. The lipid bilayer membrane is the core carrier for the encapsulation and protection of active factors. High electron density regions are visible inside some vesicles (possibly loading sites for pungent glycosides), indicating successful encapsulation. Particle size consistency: consistent with... Figure 3 The DLS data matching was used to further verify the reliability of the preparation process.

[0058] like Figure 5As shown, control represents cells living in PBS buffer; SerMA represents cells living in a hydrogel polymerized from methacrylated sericin (sericin bio-ink); PUN8@SerMA-mCMVs represents cells living in a hydrogel polymerized from cell capsule bio-ink (PUN8@SerMA-mCMVs) with antioxidant activity. (A) Fluorescence detection diagram (the stronger the green fluorescence, the weaker the antioxidant capacity); (B) ROS scavenging rate.

[0059] Results Display: Fluorescence intensity: The green fluorescence of the PUN8@SerMA-mCMVs group was significantly weaker than that of the Control and SerMA groups. Figure 5 A) indicates that it has a lower ROS level and stronger antioxidant capacity. The sustained-release properties of the cell capsule allow pungent glycosides to maintain their activity for a long time and continuously scavenge ROS.

[0060] Quantitative data: The ROS clearance rate in the PUN8@SerMA-mCMVs group was significantly higher than that in the control group ( Figure 5 (B) This demonstrates that the sustained release of pungent glycosides effectively reduces cellular oxidative stress. Vesicles protect pungent glycosides from degradation and deliver them specifically to chondrocytes, ensuring their antioxidant efficacy.

[0061] like Figure 6 As shown, the appearance and live cell staining images of the sample obtained by DLP printing are as follows: (A) Appearance and live cell staining images of the printed sample, with live cells showing green fluorescence; (B) Bar chart of cell activity and cell proliferation capacity of the sample obtained by 3D bioprinting.

[0062] Figure 6 a: The printed sample has a complete structure and complex geometry (such as a mesh or porous structure), proving that the ink is compatible with the DLP printing process. The uniform distribution of green fluorescence (such as Calcein-AM labeling) indicates high cell viability and uniform dispersion.

[0063] Figure 6 b: Initial activity: viable cell rate >95% after printing; proliferation capacity: cell number increased significantly after 7 days, proving that the bio-ink supports cell growth.

[0064] Figures 3-6 The innovative aspects of this invention—a cell capsule bio-ink that combines targeted sustained release, printability, and bioactivity—are supported by structural characterization (DLS, TEM), functional verification (antioxidant properties, cell activity), and application demonstration (3D printing). These data provide solid evidence for the technical effectiveness and industrial application of this invention.

[0065] The biological principles underlying this invention are as follows: Cell capsules are cell membrane vesicles loaded with bio-ink. These cell membrane vesicles are used to encapsulate active factors or growth factors. At the same time, acrylate-polyethylene glycol-succinimide esters are reacted with the cell membrane vesicles to obtain a large number of acrylate-grafted cell membrane vesicles. Such cell membrane vesicles carrying active factors / drugs / growth factors are called cell capsules.

[0066] These cell membrane vesicles can be photopolymerized with commonly used methacrylated polymers, such as sericin and gelatin, under the initiation of a photoinitiator to form hydrogels.

[0067] This hydrogel, because cell membrane vesicles are copolymerized with polymer materials, prevents the free movement of cell membrane vesicles carrying active or growth factors. However, in existing technologies, enzymes released by cells during growth gradually degrade the hydrogel scaffold, causing the cell membrane vesicles to be phagocytosed by the cells. In this invention, because the cell membrane vesicles and the cell membranes of the loaded cells are of the same type, it achieves a certain degree of targeting. Simultaneously, the cells grow gradually, and the cell membrane vesicles are gradually phagocytosed, resulting in a slow-release effect. The cell membrane vesicles separate the external environment from the internal environment, can carry various lipid-soluble factors, and can also ensure the stable existence of active factors such as protein growth factors and mRNA without degradation.

[0068] The present invention has been described above by way of example with reference to the accompanying drawings. Obviously, the implementation of the present invention is not limited to the above-described manner. Any improvements made using the inventive concept and technical solution of the present invention, or the direct application of the inventive concept and technical solution of the present invention to other occasions without modification, are all within the protection scope of the present invention.

Claims

1. A method for preparing cell capsule bio-ink, comprising the following steps: Step 1: Prepare acrylate-modified cell membrane vesicles (mCMVs) using cell membrane vesicles and acrylate-polyethylene glycol-succinimide (AC-PEG-NHS). Step 2: Using the acrylate-modified cell membrane vesicles (mCMVs) obtained in Step 1 and active factors, cell capsules loaded with active factors (PUN@mCMVs) are prepared. Step 3: Mix the methacrylated polymer material, photoinitiator, live cells, and cell capsules (PUN@mCMVs) loaded with active factors obtained in Step 2 to form a photocrosslinkable cell capsule bio-ink.

2. The method for preparing cell capsule bio-ink according to claim 1, characterized in that: In step 1, cell membrane vesicles are mixed with acrylate-polyethylene glycol-succinimide ester (AC-PEG-NHS) and prepared by light-protected reaction to acrylate-modified cell membrane vesicles (mCMVs).

3. The method for preparing cell capsule bio-ink according to claim 2, characterized in that: The cell membrane vesicles mentioned in step 1 are derived from mammalian cell membranes, preferably cell membrane vesicles of the same type as the cells they carry.

4. The method for preparing cell capsule bio-ink according to claim 2, characterized in that: The reaction conditions in step 1 include: the reaction is carried out in PBS containing the photoinitiator LAP.

5. The method for preparing cell capsule bio-ink according to claim 1, characterized in that: Step 2: The acrylate-modified cell membrane vesicles (mCMVs) obtained in Step 1 are mixed with active factors and then successively dispersed by ultrasonication and extruded by a liposome extruder. After purification by ultracentrifugation, cell capsules loaded with active factors (PUN@mCMVs) are finally obtained.

6. The method for preparing cell capsule bio-ink according to claim 5, characterized in that: The liposome extruder described in step 2 uses a porous membrane with decreasing pore size for stepwise extrusion.

7. The method for preparing cell capsule bio-ink according to claim 5, characterized in that: The active factor mentioned in step 2 is at least one of a lipid-soluble active factor, a growth factor, a drug, or a functional RNA, preferably punicin (PUN).

8. The method for preparing cell capsule bio-ink according to claim 1, characterized in that: The methacrylated polymer material mentioned in step 3 is at least one of methacrylated sericin (SerMA) and methacrylated gelatin.

9. The method for preparing cell capsule bio-ink according to claim 1, characterized in that: The living cells mentioned in step 3 are chondrocytes.

10. A cell capsule bio-ink, characterized in that: The invention comprises cell capsules carrying active factors (PUN@mCMVs), methacrylated polymer materials, photoinitiators, and live cells, wherein the cell capsules are internally loaded with active factors and have photocrosslinking properties.

11. The cell capsule bio-ink according to claim 10, characterized in that: The lipid bilayer membrane of the cell capsule isolates the internal active factors from the external hydrogel environment, preventing the degradation of the active factors.

12. The application of the cell capsule bio-ink according to claim 10 or 11, characterized in that: Used for 3D bioprinting, it forms a hydrogel scaffold through photopolymerization to achieve targeted and sustained release of active factors.

13. The application of the cell capsule bio-ink according to claim 12, characterized in that: The 3D bioprinting is digital light processing (DLP) printing, with printing parameters including a light intensity of 10-50 mW / cm². 2 The exposure time is 5-30 seconds.

14. The application of the cell capsule bio-ink according to claim 12, characterized in that: The hydrogel scaffold gradually releases active factors through enzymatic degradation during cell growth, and the cell capsule is homologous to the cell membrane of the loaded cells, thus achieving targeted delivery.

15. The application of the cell capsule bio-ink according to claim 10 or 11, characterized in that: Used for 3D bioprinting, hydrogel scaffolds are formed through photopolymerization, and these scaffolds are implanted into the body to become artificial tissues or organs.

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