Cell orientation regulation bio-3d printing method based on temperature control interface fusion and application

Abstract

This invention relates to a cell orientation-controlled bio-3D printing method and its application based on temperature-controlled interface fusion, comprising: 1. Preparing a composite bio-ink containing gelatin and fibrin precursors; 2. Low-temperature deposition molding: controlling the temperature of the printing nozzle to room temperature to ensure the fluidity of the composite bio-ink, and controlling the temperature of the receiving substrate, receiving base, or mold to low temperature; depositing the composite bio-ink on the receiving substrate, relying on the low-temperature gelation of gelatin to maintain the morphology of the printed lines, perfusion layers, or multilayer structures to obtain a three-dimensional structure; 3. Fibrin cross-linking and curing: applying thrombin solution to the three-dimensional structure to cause the fibrin precursors to cross-link and form a stable fibrin network; this invention can achieve random orientation growth of cells inside the hydrogel or directional growth along the printing path by selecting whether to perform short-term temperature rise interface fusion treatment; this invention can be used to construct tumor microenvironment chips, immune co-culture chips, vascularized tissue chips, and directional tissue constructs.

Description

Technical Field

[0001] This invention belongs to the field of bio-3D printing and biomimetic cell microenvironment construction technology, specifically involving a cell orientation regulation bio-3D printing method and its application based on temperature-controlled interface fusion. Background Technology

[0002] Bioprinting technology can deposit cells, biomaterials, and bioactive factors according to a pre-defined spatial structure, making it an important technology for constructing living tissues, disease models, and organ-on-a-chip. For tumor microenvironment chips, immune co-culture chips, vascularized tissue chips, and directional tissue constructs, the ability to controllably regulate cell spatial distribution and orientation behavior within a three-dimensional hydrogel is a key factor affecting the realism and functionality of the model.

[0003] Fibrin is a commonly used natural extracellular matrix mimicry material with good cell compatibility, supporting the adhesion, spreading, and migration of various cells. However, when using fibrin precursor solutions alone for printing, their viscosity and molding ability are often insufficient, making it difficult to stably maintain the printed structure. Gelatin, on the other hand, is temperature-responsive, forming a gel at low temperatures to aid molding, and undergoing sol-gelation at room temperature or 37°C. Therefore, it is often used to improve the printing stability of bio-inks.

[0004] However, when using gelatin-containing composite bio-inks for multilayer or sandwich structure printing, the printed lines, interlayer interfaces, or material boundaries formed during the cryogenic molding process may remain within the hydrogel, constraining cell spreading and migration directions. For the construction of oriented tissues such as myocardium, muscle, and nerves, this printing path-induced cell orientation may have a positive effect; however, for tumor microenvironment chips, immune cell infiltration models, and blood vessel-tumor interaction models, if cells are restricted by the printing path and grow in a directional manner, it may introduce artificial structural bias, hindering the accurate evaluation of cell-cell interactions, migration behavior, and drug responses.

[0005] Therefore, there is an urgent need for a method that can regulate cell orientation behavior according to different application requirements based on the same bio-ink system and printing process: when it is necessary to construct random three-dimensional microenvironments, weaken or eliminate the constraint of the printing path on the cell spreading direction; when it is necessary to construct oriented tissue structures, retain the inductive effect of the printing path or interlayer interface on cells, so as to solve the above technical problems. Summary of the Invention

[0006] This invention provides the following technical solution: a cell orientation-controlled bio-3D printing method based on temperature-controlled interface fusion, comprising the following steps: Step S1: Preparation of composite bio-ink: The composite bio-ink contains gelatin and fibrin precursor; the fibrin precursor can be cross-linked under the action of thrombin to form a three-dimensional fibrin network.

[0007] Step S2, Low-temperature deposition molding: The temperature of the printhead is controlled at 15-30 ℃ to ensure the fluidity of the composite bio-ink, and the temperature of the receiving plate, receiving substrate or mold is controlled at 0-10 ℃; the composite bio-ink is deposited on the receiving plate by pouring or printing, and the printing lines, pouring layers or multi-layer structure morphology is maintained by the low-temperature gelation of gelatin to obtain a three-dimensional structure.

[0008] Step S3, fibrin cross-linking and solidification: Thrombin solution is applied to the three-dimensional structure to cross-link the fibrin precursor and form a stable fibrin network.

[0009] Preferably, the step between step S2 and step S3 further includes: Step S2.5, Short-time heating interface fusion treatment: Before the fibrin precursor is fully cross-linked, the three-dimensional structure is subjected to short-time heating treatment; short-time heating causes partial solidification of gelatin, weakens the interface boundary between the printing layer and the printing line, and realizes interlayer interface fusion; after heating, the three-dimensional structure is cooled and re-coagulated to stabilize the overall structure.

[0010] The dual-mode regulation of cell orientation is achieved by whether or not the short-term heating treatment in step S2.5 is performed: when the short-term heating treatment is performed, the interface fusion eliminates the printing path constraint, and the cells spread and migrate in a random orientation; when the short-term heating treatment is omitted, the printing interface boundary is preserved, and the cells spread and migrate in a directional manner along the printing path; the random orientation growth or directional orientation growth of cells in the three-dimensional structure is regulated.

[0011] During short-time heating, the interface boundaries between adjacent printed layers, adjacent printed lines, or adjacent perfusion layers are weakened or merged, causing cells to spread and migrate in a random orientation within the three-dimensional structure. Without short-time heating, or when the heating is insufficient to cause interface fusion between adjacent printed layers, adjacent printed lines, or adjacent perfusion layers, the printing path boundaries or interlayer boundaries are preserved in the three-dimensional structure, allowing cells to spread and migrate oriented along the printing path.

[0012] More preferably, in step S1, the gelatin concentration in the composite bio-ink is 1%–10% by volume; the fibrin precursor concentration is 0.05%–2% by volume; and the fibrin precursor is fibrinogen. The fibrin precursor forms a fibrin network after being cross-linked with thrombin.

[0013] More preferably, in step S2, the temperature of the printing nozzle is controlled at 20-28°C; the temperature of the receiving substrate is 2-6°C; and the gelatin is allowed to stand at a low temperature for 4-6 minutes after deposition to allow it to fully gel and solidify.

[0014] More preferably, the short-time heating conditions in step S2.5 are: temperature 25-40℃; treatment time 0.5-5min.

[0015] More preferably, in step S3, the thrombin solution concentration is 1–100 U / mL; the cross-linking is divided into two stages: cross-linking at 3–5°C for 10–20 min, followed by cross-linking at 36–38°C for 30–50 min. Preferably, the low-temperature cross-linking temperature is 4°C and the high-temperature cross-linking temperature is 37°C. More preferably, the three-dimensional structure is a sandwich structure, comprising, in sequence, a bottom hydrogel layer, a cell printing layer, and a covering hydrogel layer.

[0016] More preferably, step S2 employs electrostatic assisted extrusion printing: a voltage is applied between the print head and the receiving substrate to cause the ink to form an electrostatic traction jet or Taylor cone, refining the deposition linewidth and achieving single-cell sequential arrangement; printing parameters: receiving distance 20-200μm, printing speed 5-30mm / s, printing air pressure 10-100Pa, applied voltage 500-2000V.

[0017] More preferably, the composite bio-ink contains a mixture of loaded cells; the cells are selected from one or more of tumor cells, immune cells, endothelial cells, stromal cells, cardiomyocytes, skeletal muscle cells, and nerve cells; the cell density is 5 × 10⁻⁶. 5 –2×10 6 cells / mL.

[0018] The beneficial effects of this invention are: 1. This invention not only utilizes the gelling properties of gelatin at low temperatures to improve the temporary molding ability of fibrin precursor composite ink, but also further utilizes the partial sol-gelling behavior of gelatin under short-term heating conditions to achieve the control of the internal interface state of the printed structure.

[0019] 2. The present invention introduces a short-time heating interface fusion step before fibrin crosslinking, which weakens or fuses the interface boundary between the printed layer, printed lines or perfusion layer, thereby eliminating or reducing the artificial constraint of the printing path on the cell spreading direction.

[0020] 3. This invention enables the switching between random and directional cell orientation modes within the same material system by performing short-term heating treatment. It is suitable for constructing tumor microenvironment chips, immune co-culture chips, and vascularized tissue chips, as well as for constructing directional myocardial, muscle, and nerve tissue structures.

[0021] 4. This invention uses a fibrin network as the main three-dimensional extracellular matrix, which has good cell compatibility and is beneficial for cell survival, spreading, migration and intercellular interaction studies.

[0022] 5. This invention can be combined with electrostatic assisted extrusion printing to achieve fine deposition of cell-loaded hydrogel lines and sequential arrangement of single cells, thereby improving the spatial accuracy of constructing active tissue and organ-on-a-chip. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the cell orientation regulation bio-3D printing method and its application based on temperature-controlled interface fusion, as described in this invention. Figure 2 This is a schematic diagram of the state changes of the fibrin precursor-gelatin composite bio-ink in the present invention during the processes of low-temperature molding, short-time heating interface fusion, and thrombin cross-linking. Figure 3 This is a schematic diagram illustrating the sandwich structure constructed according to the present invention; Figure 4 This is a schematic diagram illustrating how the printing path or interlayer boundary is preserved and cells are induced to spread in a directional manner when the present invention does not undergo short-term heating treatment; Figure 5 This is a schematic diagram illustrating how the interlayer interface fuses and induces random orientation spreading of cells after a short-term heating treatment according to the present invention. Detailed Implementation

[0024] The relevant technologies of this invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0025] like Figures 1-5 As shown, the cell orientation-controlled bio-3D printing method based on temperature-controlled interface fusion in this embodiment includes the following steps: First, a composite bio-ink containing fibrin precursor and gelatin is prepared. The fibrin precursor is preferably fibrinogen, which can cross-link to form a fibrin network under the action of thrombin; gelatin is used to provide temporary gelling and shaping capabilities in a low-temperature environment after deposition.

[0026] Secondly, the printhead is kept at room temperature or near room temperature, preferably 25°C, to maintain good flowability and extrudability of the composite bio-ink; at the same time, the receiving plate, receiving substrate or mold is kept at low temperature, preferably 4°C, to allow the deposited composite bio-ink to cool rapidly and maintain the morphology of the printed lines, infusion layers or multi-layer structures by relying on gelatin gelation.

[0027] Furthermore, the three-dimensional structure is subjected to a short-term heating treatment before the fibrin precursor is fully cross-linked. This short-term heating treatment causes partial solubilization of the gelatin, weakens the interfacial boundaries between adjacent printed layers, adjacent printed lines, or adjacent infusion layers, and promotes interfacial fusion between multilayer structures or printed lines.

[0028] Subsequently, the three-dimensional structure was cooled to allow the gelatin to regel and maintain its overall shape; then thrombin solution was added to crosslink the fibrin precursors and form a stable fibrin network.

[0029] By selecting whether to perform a short-term heating treatment, two different cell orientation regulation modes can be obtained: when a short-term heating treatment is performed, the printing path boundary or interlayer boundary is weakened, and the cells spread and migrate randomly in the continuous three-dimensional hydrogel matrix; when a short-term heating treatment is not performed, the printing path boundary or interlayer boundary is preserved, and the cells spread and migrate oriented along the printing path direction.

[0030] Preferably, the mass-volume percentage concentration of gelatin in the composite bio-ink is 1%–10%, more preferably 3.5%; and the mass-volume percentage concentration of fibrin precursor is 0.05%–2%, more preferably 0.3%.

[0031] Preferably, the short-time heating treatment temperature is 25–40 °C, more preferably 37 °C; the treatment time is 0.5–5 min, more preferably 1.5 min.

[0032] Preferably, the concentration of the thrombin solution is 1–100 U / mL, more preferably 10 U / mL; the thrombin crosslinking includes low-temperature crosslinking at 4 °C for 10–20 min and crosslinking at 37 °C for 30–50 min.

[0033] Preferably, the three-dimensional structure is a sandwich structure, comprising a bottom hydrogel layer, a cell printing layer, and a covering hydrogel layer.

[0034] Preferably, the method can also be combined with electrostatic assisted extrusion printing, where a voltage is applied between the printhead and the receiving substrate, causing the composite bio-ink to form an electrostatic traction jet or Taylor cone at the printhead end, reducing the hydrogel deposition linewidth and achieving single-cell sequential arrangement.

[0035] Example Example 1: Short-term heating interface fusion to construct a three-dimensional hydrogel structure with randomly oriented cells: This embodiment provides a method for constructing a three-dimensional hydrogel structure with random cell orientation through interface fusion by short-time heating.

[0036] First, a composite bio-ink containing 3.5% gelatin and 0.3% fibrin precursor was formulated. Percentages are by weight / volume. The preferred fibrin precursor is fibrinogen, which forms a fibrin network after cross-linking with thrombin.

[0037] Then, 200 μL of composite bio-ink was poured into the bottom of the mold to form the bottom hydrogel layer. The mold was then placed at 4 ℃ for 5 min to allow the gelatin to gel and maintain the bottom layer morphology.

[0038] Subsequently, the cells were mixed with the composite bio-ink to obtain a cell-containing composite bio-ink. The cell density was 5 × 10⁻⁶. 5 –2×10 6 cells / mL. Cell-loaded lines were printed on the underlying hydrogel layer using electrostatic assisted extrusion printing. During printing, the printhead temperature was room temperature, preferably 25 °C, to maintain the extrudability of the composite bio-ink; the receiving plate temperature was 4 °C, allowing the deposited composite bio-ink to cool rapidly and maintain the line morphology through gelatin gelation.

[0039] The printing parameters are: receiving distance 50 μm, printing speed 10 mm / s, printing air pressure 50 Pa, and applied voltage 1000V.

[0040] After printing, the printed structure was stored at 4 °C for 5 min. Then, 200 μL of composite bio-ink was applied to the printed structure to form a covering hydrogel layer, and the structure was stored at 4 °C for 5 min again to obtain a sandwich structure consisting of a bottom hydrogel layer, a cell printing layer, and a covering hydrogel layer.

[0041] Next, the sandwich structure was incubated at 37 °C for 1.5 min to partially solubilize the gelatin, weakening the interfacial boundaries between the bottom hydrogel layer, the cell-printed layer, and the overlay hydrogel layer, thus promoting interfacial fusion of the three layers. After a short heating treatment, the sandwich structure was again stored at 4 °C for 5 min to allow the gelatin to regel and stabilize the structure.

[0042] Subsequently, 10 U / mL thrombin solution was added, and cross-linking was first performed at 4 ℃ for 10–20 min, followed by cross-linking at 37 ℃ for 30–50 min, to allow the fibrin precursor to cross-link and form a stable fibrin network. After cross-linking was completed, the cross-linking solution was removed and added to complete culture medium for incubation.

[0043] In the three-dimensional hydrogel structure prepared using this method, the short-time heating treatment promotes interlayer interface fusion, and cells are no longer primarily constrained by the printing path boundaries within the continuous fibrin matrix, enabling them to spread and migrate in random orientations. This structure is suitable for constructing tumor microenvironment chips, immune co-culture chips, and vascularized tissue chips.

[0044] Example 2: Construction of a cell-oriented three-dimensional hydrogel structure by omitting short-term heating treatment: This embodiment provides a method for constructing cell-oriented three-dimensional hydrogel structures by preserving the boundaries of the printing path.

[0045] The difference between this embodiment and Embodiment 1 is that after constructing the sandwich structure, the interface fusion treatment with a short heating at 37 ℃ is not performed, but thrombin cross-linking is performed directly.

[0046] Specifically, 200 μL of a composite bio-ink containing 3.5% gelatin and 0.3% fibrin precursor was poured into the bottom of the mold and stored at 4 °C for 5 min. Subsequently, cell-containing composite bio-ink lines were printed onto the bottom hydrogel layer at a cell density of 5 × 10⁻⁶ cells / cm². 5 –2×10 6 cells / mL. After printing, store at 4 ℃ for 5 min. Then cover with 200 μL of composite bio-ink and store at 4 ℃ for 5 min.

[0047] Subsequently, without a short-term heating treatment at 37 °C, 10 U / mL thrombin solution was directly added, and cross-linking was first performed at 4 °C for 10–20 min, followed by cross-linking at 37 °C for 30–50 min. After cross-linking was completed, the cross-linking solution was removed and cultured in complete culture medium.

[0048] Because short-term heating for interface fusion was not performed, the printed line boundaries and interlayer interfaces were partially preserved within the 3D hydrogel. Cells, constrained by the printed path boundaries during spreading and migration, tend to grow directionally along the printed lines. This method can be used to construct directional myocardial tissue, muscle tissue, nerve tissue, or other biomimetic tissue structures requiring orderly cell arrangement.

[0049] Example 3: Electrostatic Assisted Extrusion Printing for Single-Cell Sequential Arrangement: This embodiment provides a method for achieving single-cell sequential arrangement by combining electrostatic assisted extrusion printing.

[0050] Cells were mixed with a composite bio-ink containing 3.5% gelatin and 0.3% fibrin precursor, at a cell density of 5 × 10⁻⁶. 5 –2×10 6cells / mL. The cell-containing bio-ink was loaded into a low-temperature printhead, and the printhead temperature was maintained at around 4 °C.

[0051] During printing, a voltage of 1000 V is applied between the printhead and the receiving substrate, the receiving distance is 50 μm, the printing speed is 10 mm / s, and the printing air pressure is 50 Pa. Under the action of the electric field, the composite bio-ink forms an electrostatic traction jet or Taylor cone at the printhead end, which refines the deposited hydrogel lines to near the single-cell scale, thereby constraining the cells to arrange themselves in a single-cell sequence along the printing line direction.

[0052] After printing, post-processing methods can be selected according to application requirements. When it is necessary to retain the sequential or oriented arrangement of single cells, no short-term heating treatment is required; thrombin solution is added directly for cross-linking. When it is necessary to make the cells spread randomly in subsequent culture, a short-term heating treatment at 37 ℃ for 1.5 min is performed first to allow the printed lines to fuse with the surrounding hydrogel, and then thrombin cross-linking is performed.

[0053] This embodiment illustrates that the present invention can not only regulate the subsequent spreading tendency of cells, but also achieve high-precision control of the initial spatial position of cells by combining electrostatic assisted printing.

[0054] Example 4: Construction of a multicellular tumor microenvironment chip: This embodiment provides a method for constructing a tumor microenvironment chip for co-culturing tumor cells with immune cells or vascular-associated cells.

[0055] First, a bottom hydrogel layer is prepared according to the method in Example 1. Then, using a dual-nozzle or multi-nozzle printing method, composite bio-inks containing tumor cells and composite bio-inks containing immune cells, endothelial cells, or stromal cells are printed onto the bottom hydrogel layer. The different cell printing areas can be arranged in parallel, adjacent, staggered, surrounding, or spatially distributed with a preset spacing.

[0056] After printing, the structure was stored at 4 °C for 5 min. Then, it was covered with 200 μL of composite bio-ink and stored at 4 °C for another 5 min to form a sandwich structure containing different cellular regions.

[0057] The sandwich structure was then incubated at 37 °C for 1.5 min to allow interfacial fusion of the bottom hydrogel layer, the cell-printed layer, and the overlay hydrogel layer. It was then stored at 4 °C for 5 min, and 10 U / mL thrombin solution was added. Crosslinking was first performed at 4 °C for 10–20 min, followed by crosslinking at 37 °C for 30–50 min. After crosslinking was complete, the crosslinking solution was removed, and complete culture medium was added for further cultivation.

[0058] Because short-time heating leads to interfacial fusion, a continuous fibrin matrix forms within the three-dimensional structure, the migration and interactions between tumor cells and immune cells, endothelial cells, or stromal cells are no longer primarily constrained by the printed path boundaries. Therefore, this chip can be used to study tumor cell migration, immune cell infiltration, tumor cell-immune cell interactions, tumor cell-vascular cell interactions, drug screening, and personalized drug evaluation.

[0059] To verify the effect of cell orientation regulation, cells were subjected to fluorescence staining and microscopic imaging after culture to extract the long axis direction, cytoskeleton direction, or migration trajectory direction, and the angular distribution, orientation index, or angular dispersion of cell orientation were calculated. Compared with the structure without short-term heating at 37 °C, the cell orientation distribution in the structure treated at 37 °C for 1.5 min was closer to a random distribution, indicating that short-term heating interface fusion can effectively weaken the directional growth induced by the printing path.

[0060] In summary, this invention provides a cell orientation-controlled bioprinting method based on temperature-controlled interface fusion. By regulating the sol-gel transition of gelatin, the interface fusion state is controlled, thereby flexibly regulating the orientation of cells in a three-dimensional matrix. This invention is simple to operate and highly compatible, enabling both the construction of ordered functional tissues with oriented cell orientation and the creation of randomly oriented three-dimensional microenvironments through interface fusion. It can be used to construct tissue-on-a-chip and organ-on-a-chip for cell-cell interaction research, showing broad application prospects in tissue engineering, regenerative medicine, and life science research.

[0061] It should be emphasized that the above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A cell orientation-controlled bio-3D printing method based on temperature-controlled interface fusion, characterized in that, Includes the following steps: Step S1: Preparation of composite bio-ink: The composite bio-ink contains gelatin and fibrin precursor; the fibrin precursor can be cross-linked under the action of thrombin to form a three-dimensional fibrin network; Step S2, Low-temperature deposition molding: The temperature of the printing nozzle is controlled at 15-30 ℃ to ensure the fluidity of the composite bio-ink, and the temperature of the receiving plate, receiving substrate or mold is controlled at 0-10 ℃; the composite bio-ink is deposited on the receiving plate by pouring or printing, and the printing lines, pouring layers or multi-layer structure morphology is maintained by the low-temperature gelation of gelatin to obtain a three-dimensional structure. Step S3, fibrin cross-linking and solidification: Thrombin solution is applied to the three-dimensional structure to cross-link the fibrin precursor and form a stable fibrin network.

2. The cell orientation-controlled bio-3D printing method based on temperature-controlled interface fusion according to claim 1, characterized in that, The step between step S2 and step S3 also includes: Step S2.5, Short-time heating interface fusion treatment: Before the fibrin precursor is fully cross-linked, the three-dimensional structure is subjected to short-time heating treatment; the short-time heating causes the gelatin to partially solubilize, weakening the interface boundary between the printing layer and the printing line, and realizing interlayer interface fusion; after heating, the three-dimensional structure is cooled and re-coagulated to stabilize the overall structure.

3. The cell orientation-controlled bio-3D printing method based on temperature-controlled interface fusion according to claim 2, characterized in that, In step S1, the gelatin mass-volume concentration in the composite bio-ink is 1% to 10%; the fibrin precursor mass-volume concentration is 0.05% to 2%; and the fibrin precursor is fibrinogen.

4. The cell orientation-controlled bio-3D printing method based on temperature-controlled interface fusion according to claim 2, characterized in that, In step S2, the temperature of the printing nozzle is controlled at 20-28 ℃; the temperature of the receiving substrate is 2-6 ℃; and the gelatin is allowed to stand at a low temperature for 4-6 minutes after deposition to allow it to fully gel and solidify.

5. The cell orientation-controlled bio-3D printing method based on temperature-controlled interface fusion according to claim 2, characterized in that, The short-time heating conditions in step S2.5 are: temperature 25-40℃; treatment time 0.5-5 min.

6. The cell orientation-controlled bio-3D printing method based on temperature-controlled interface fusion according to claim 2, characterized in that, In step S3, the concentration of thrombin solution is 1-100 U / mL; the cross-linking is divided into two stages: cross-linking at 3-5℃ for 10-20 min, and then cross-linking at 36-38℃ for 30-50 min.

7. The cell orientation-controlled bio-3D printing method based on temperature-controlled interface fusion according to claim 2, characterized in that, The three-dimensional structure is a sandwich structure, consisting of a bottom hydrogel layer, a cell printing layer, and a covering hydrogel layer.

8. The cell orientation-controlled bio-3D printing method based on temperature-controlled interface fusion according to claim 2, characterized in that, In step S2, electrostatic assisted extrusion printing is used: a voltage is applied between the print head and the receiving substrate to make the ink form an electrostatic traction jet or Taylor cone, which refines the deposition linewidth and realizes the sequential arrangement of single cells; printing parameters: receiving distance 20-200μm, printing speed 5-30mm / s, printing air pressure 10-100Pa, applied voltage 500-2000V.

9. The cell orientation-controlled bio-3D printing method based on temperature-controlled interface fusion according to claim 2, characterized in that, The composite bio-ink contains a mixture of loaded cells; the cells are selected from one or more of tumor cells, immune cells, endothelial cells, stromal cells, cardiomyocytes, skeletal muscle cells, and nerve cells; the cell density is 5 × 10⁻⁶. 5 –2×10 6 cells / mL.

10. The application of the cell orientation-controlled bio-3D printing method based on temperature-controlled interface fusion according to any one of claims 1 to 9 in the preparation of cell orientation-controlled three-dimensional hydrogel constructs and the preparation of tumor microenvironment chips.

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