Microfluidically controlled 3D printed hydrogels using decellularized fish liver matrix, their fabrication method, and applications.

Abstract

This invention provides a microfluidically controlled 3D printed hydrogel using a decellularized fish liver matrix for liver regeneration, and a method for producing the same. The decellularized fish liver matrix (dECM) is combined with gelatin methacryloyl (GelMA) to support induced pluripotent stem cell-derived hepatocytes (iPSC-hep) for liver regeneration. The microfluidically controlled 3D printed hydrogel using the decellularized fish liver matrix of this invention exhibits excellent biocompatibility, retains complete endogenous cell growth factors, maintains cellular physiological activity, ensures effective cell encapsulation, and is advantageous for the stable functional expression of iPSC-hep. After transplantation into mice with acute liver failure, survival rate and liver function significantly improved, and liver regeneration and repair were promoted.

Description

Technical Field

[0001] The present invention relates to the field of biomedical materials, and specifically to a microfluidic control 3D printed hydrogel using a decellularized matrix of fish liver for liver regeneration and a method for producing the same.

Background Art

[0002] Liver transplantation is an effective method for the treatment of end-stage liver diseases, but the scarcity of liver donors has limited its widespread clinical application. Tissue engineering is considered a promising alternative to liver donors due to its ability to construct functional liver tissue. Hydrogels are advanced materials for creating various structures required for liver transplantation and have good biocompatibility and cell proliferation ability for hepatocytes derived from induced pluripotent stem cells (iPSC-heps). However, these hydrogels generally have limited physiological activity expression and are clearly different from the composition of natural liver tissue. In addition, most hydrogels require complex manufacturing processes, leading to potential biosafety problems and reduced therapeutic effects. Therefore, the need for innovative hydrogels with inherent physiological activity and sufficient support for liver function remains unmet.

[0003] Decellularized fish liver is a type of natural material with excellent biocompatibility and contains complete endogenous cell growth factors, including collagen and glycosaminoglycans. Fish liver from aquatic sources is more abundant and less expensive than livers from terrestrial animals. Microfluidic technology is recognized for its precise control of fluid dynamics and is one of the advanced forms used in additive manufacturing such as drug delivery, cell culture, and biosensors. 3D printing enables the custom design of implantable hydrogels to meet specific clinical needs. By leveraging these advantages and integrating microfluidic and 3D printing technologies, it becomes possible to manufacture custom-made liver regeneration hydrogels. Therefore, it is highly desirable to develop a new type of scaffold that effectively promotes liver repair by combining decellularized fish liver hydrogel as a Bio-link with microfluidic-assisted 3D printing technology. [Overview of the project] [Problems that the invention aims to solve]

[0004] Objective of the present invention: The present invention aims to solve the above technical problems, and we provide a hydrogel made of a decellularized fish liver matrix necessary for liver regeneration, which is fabricated using a simple microfluidic 3D printing technology and used for liver repair by orthotopic transplantation. This hydrogel is prepared by a method using microfluidic 3D printing technology, and the method is simple, versatile, and easy to mass-produce. [Means for solving the problem]

[0005] Technical solution: A mixed material hydrogel scaffold fabricated using microfluidic controlled 3D printing technology, using a decellularized fish liver matrix combined with gelatin methacryloyl as a scaffold and human induced pluripotent stem cell-derived hepatocytes as the cell source. This is a microfluidic controlled 3D printed hydrogel using a decellularized fish liver matrix.

[0006] The procedure for fabricating a microfluidically controlled 3D-printed hydrogel using a decellularized fish liver matrix is ​​as follows: 1) Production of hepatocytes derived from human induced pluripotent stem cells, 2) Preparation of a decellularized matrix from fish liver, 3) A 3D scaffold model is designed, and gelatin methacryloyl, the decellularized fish liver matrix prepared in step 2), human induced pluripotent stem cell-derived hepatocytes prepared in step (1), and a photoinitiator are thoroughly mixed to form a bio-link, which is then solidified into a scaffold that maintains its shape under ultraviolet irradiation.

[0007] The specific procedure in 2) above is: a. Prepare fresh fish liver and complete blood vessels. b. Decellularization is performed by continuously perfusing fish liver with a surfactant solution at room temperature. c. Wash the material obtained in step b with phosphate-buffered saline. d. Dissolve the decellularized fish liver matrix obtained in step c above in an aqueous acetic acid solution containing pepsin.

[0008] Preferably, the photoinitiator is 2-hydroxy-2-methyl-1phenyl-1propanone.

[0009] Preferably, the surfactant is sodium dodecyl sulfate or sodium deoxycholate.

[0010] Preferably, the concentration of the surfactant solution used in step b is 1% (g / 100mL, i.e., 1 g of surfactant dissolved in 100 mL of water), the rate of continuous perfusion of the fish liver is 10 mL / min, and the duration is 3 hours.

[0011] Preferably, the concentration of pepsin dissolved in the aqueous acetic acid solution containing pepsin in step d is 1% (g / 100mL, i.e., 1 g of surfactant dissolved in 100 mL of water), and the concentration of acetic acid is 100 mM.

[0012] Preferably, the mass ratio in the reaction system of the decellularized fish liver matrix, gelatin methacryloyl, and photoinitiator is 1.5 to 3:7.5:1.

[0013] The present invention also provides applications for the preparation of liver regeneration drugs using microfluidically controlled 3D printed hydrogels based on a decellularized fish liver matrix. [Effects of the Invention]

[0014] The beneficial effects are as follows:

[0015] (1) The microfluidically controlled 3D printed hydrogel using a decellularized fish liver matrix designed by the present invention for liver regeneration has excellent biocompatibility.

[0016] (2) The hydrogel made from a decellularized fish liver matrix used for liver regeneration provided by the present invention is prepared by a method using microfluidic controlled 3D printing technology, and is easy to operate, highly reproducible, requires little technical skill, is highly versatile, highly adaptable, and easy to mass-produce.

[0017] (3) The microfluidically controlled 3D printed hydrogel using a decellularized fish liver matrix produced by the present invention for liver regeneration retains a unique fibrous structure in which supporting cells adhere and migrate, maintaining the physiological activity of cells, ensuring effective cell encapsulation, and is advantageous for the stable functional expression of iPSC-hep. After transplantation into mice with acute liver failure, survival rate and liver function were significantly improved, and liver regeneration and repair were promoted. [Brief explanation of the drawing]

[0018] [Figure 1]This is a conceptual diagram of the preparation and evaluation of a decellularized fish liver matrix (dECM). Figure A shows the effect of 1% SDS on the decellularization time of fish liver at 15, 30, 120, 240, and 360 minutes, respectively, with a scale of 0.5 cm. Figure B shows the DNA content in protean and decellularized fish liver. Figure C shows scanning electron microscope images, H&E stained images, and DAPI stained images of protean and decellularized fish liver. The scales are 100 μm (i), 20 μm (ii, iii, iv), and 50 μm (v, vi), respectively. Figure D shows immunofluorescence staining of laminin, type I collagen, and type IV collagen in protean and decellularized fish liver, with DAPI staining showing the cell nucleus, with a scale of 100 μm. Figure E shows the DAPI quantitative fluorescence analysis of group D, indicating that the cell nucleus was removed after decellularization. [Figure 2] This is a conceptual diagram of the bioprinting characteristics evaluation of hydrogels using dECM. Figure A shows the fabrication of a hydrogel mixed with dECM and GelMA using microfluidic controlled 3D printing. Figure B is a conceptual diagram of the scaffolds for the second, fourth, and sixth layers using microfluidic controlled 3D bioprinting. Figures C-D are fluorescence macro images (C) and close-up images (D) of the scaffolds for the second, fourth, and sixth layers after the addition of green fluorescent nanoparticles. Figure E is an SEM image depicting the microstructure inside the bioprint scaffold, with scales of 100 μm (i) and 50 μm (ii), respectively. Figures F-G are comparative analysis diagrams of the swelling rate (F) and degradation rate (G) of the bioink group without dECM (GelMA), the 1.5% dECM bioink group, and the 3% dECM bioink group. [Figure 3]Conceptual diagram of biocompatibility evaluation of hydrogel scaffolds by dECM and cell function maintenance. Figure A shows live / dead imaging of iPSC-heps seeded on scaffolds of GelMA, 1.5% dECM hydrogel, and 3% dECM hydrogel printing on days 1, 3, 5, and 7. The scale is 50 μm. Figure B is a conceptual diagram of cell attachment after culturing cells on a hydrogel scaffold with dECM for 7 days as shown by a scanning electron microscope. The scales are 200 μm, 100 μm, and 20 μm from top to bottom respectively. Figure C is a quantitative analysis diagram of live / dead staining after culturing cells on three types of scaffolds for 7 days. Figure D is the mRNA expression level on scaffolds of hydrogel with 1.5% dECM and hydrogel with 3% dECM. Figures E - F are fluorescence immunostaining of cells on a scaffold of 3% dECM hydrogel, and the scale is 10 μm. [Figure 4] Conceptual diagram for evaluating the therapeutic effect of treating ALF mice by transplanting a hydrogel scaffold with dECM. Figure A is a conceptual diagram of the liver transplantation of an ALF mouse model and a hydrogel scaffold. Figure B is a conceptual diagram of the hydrogel scaffold adhering to the liver. Figure C is in vivo imaging of a small animal showing that the hydrogel scaffold has settled in the liver. Figures D - F are conceptual diagrams of the survival rate (D), aspartate aminotransferase (AST) (E), and alanine aminotransferase (ALT) (F) levels of four groups of mice. Figures G - H show H&E staining (H), liver injury level, and the corresponding Suzuki Score (G), and the scale in (H) is 100 μm. [Figure 5] Conceptual diagram for evaluating the transplantation therapeutic effect of a hydrogel scaffold with dECM by immunohistochemistry. Figures A - C are fluorescence merge diagrams of representative Ki-67 (A), TUNEL (B), and CD68, Nrf2, HO-1, and DAPI (C) of liver samples in each group. Figures D - H are fluorescence quantitative analysis diagrams of Ki-67 (D), TUNEL (E), CD68 (F), Nrf2 (G), and HO-1 (H), and the scale is 100 μm.

Mode for Carrying Out the Invention

[0019] To deepen the understanding of the present invention, the present invention will be described in more detail below in conjunction with examples and drawings. The examples are only for interpreting the present invention and do not limit the protection scope of the present invention.

[0020] The human induced pluripotent stem cells (hiPSC) used in the following examples were purchased from the Hepatocyte Bank of the Chinese Academy of Sciences.

[0021] In the following examples, "concentration" means the mass of the solute in 100 mL of the solvent. For example, if the concentration of GelMA in the reaction system is 7.5%, it means that the mass of GelMA per 100 mL of the reaction solvent is 7.5 g.

Example

[0022] Preparation of a Microfluidic-Controlled 3D Printed Hydrogel Using a Decellularized Matrix of Fish Liver

[0023] (1) Preparation of human induced pluripotent stem cell-derived hepatocytes (hiPSC-hep) (Existing technical literature cited in the preparation procedure: Chen, Sitong et al. Hepatic spheroids derived from human induced pluripotent stem cells in bio-artificial liver rescue porcine acute liver failure. Cell Research, (2019)0:1-3)

[0024] Using mTeSR1 medium containing Matrigel, human induced pluripotent stem cells (hiPSC) were cultured in a 6-well plate at 37 °C under 5% CO2 conditions (5×10 , ,

[0024] , 5Cells were cultured in RPMI1640 medium containing activin A, BMP4, bFGF, B27, and Wnt3a for 1 day, then transferred to RPMI1640 medium containing activin A, BMP4, and bFGF and cultured for 3 days to promote the development of final endodermal cells. To promote hepatocyte formation, endodermal cells were cultured in RPMI1640 medium containing KGF, SB431542, and B27 for 2 days, then cultured in RPMI1640 medium containing KGF, BMP4, BMP2, bFGF, and B27 for 3 days. To promote the differentiation of hepatocytes into hepatic progenitor cells (HPCs), hepatoblasts were cultured in DMEM / F12 medium containing B27, forskolin, SB431542 EGF, CHIR99021, LPA, Dex, and S1P for 6-8 days. To generate mature hepatocytes (hiPSC-Heps), HPCs were cultured for 21 days in Williams' E medium containing B27, forskolin, and SB431542.

[0025] (2) Preparation of decellularized fish liver matrix: Complete livers and blood vessels were removed from fresh fish. Next, the livers were decellularized by continuous perfusion with sodium dodecyl sulfate (SDS) at room temperature. Subsequently, the livers were washed with phosphate-buffered saline to remove all remaining decellularizing agents. All of the above procedures were performed under sterile conditions. The prepared decellularized fish liver matrix (dECM) was dissolved in a solution containing pepsin and acetic acid. The concentration of SDS was 1%, the concentration of pepsin was 1%, and the concentration of acetic acid was 100 mM. The continuous perfusion rate was 10 mL / min, and the perfusion time was 3 hours.

[0026] (3) Fabrication of microfluidically controlled 3D printed hydrogels using a decellularized fish liver matrix

[0027] After disinfecting the microfluidic printer, a 3D scaffold model was designed and printed. The decellularized fish liver matrix prepared in step (2), GelMA, hiPSC-heps prepared in step (1), and the photoinitiator HMPP (2-hydroxy-2-methyl-1phenyl-1-propanone) were thoroughly mixed in water to form a Bio-link. It was immediately solidified into a hydrogel scaffold that retained its shape under UV irradiation. The concentrations of dECM in the reaction system were 1.5% and 3%, respectively, the concentration of GelMA in the reaction system was 7.5%, the concentration of HMPP in the reaction system was 1%, and the amount of hiPSC-heps used was 10 cells. 7 They were treated as individuals. [Examples]

[0028] Evaluation of decellularized extracellular matrix (dECM) in fish liver

[0029] 1% sodium dodecyl sulfate (SDS) was used as a decellularizing agent to promote the decellularization process via the portal circulation of fish liver. As the perfusion time was extended, the color of the entire liver changed from opaque to transparent from the center to the periphery, and the integrity of the intrahepatic biliary system and overall structure was well preserved (Figure 1A). As shown in Figure 1B, quantitative DNA analysis showed that the tissue contained less than 50 ng of double-stranded DNA per milligram. This indicates that the decellularization process effectively eluted DNA from the fresh tissue. Scanning electron microscopy (SEM) showed that, compared to the original fish liver, the unique fibrous structure in which supporting cells adhered and migrated was retained even after decellularization (Figure 1C). Furthermore, hematoxylin-eosin (H&E) staining and 4',6-diamidino-2-phenylindole (DAPI) immunofluorescence staining showed that there were no apparent residual nuclei in the decellularized liver tissue, confirming that cells in the liver were effectively removed. H&E staining revealed that even after hepatocyte structures were lysed, the liver's intrinsic systems were completely preserved. This means that the ECM and structural scaffold, which are crucial foundations for liver function, were maintained. This is extremely important for maintaining the structure and function of the organ. Based on this, we stained the ECM components that are essential for tissue regeneration. The results showed that components such as laminin, type I collagen, and type IV collagen were retained even after decellularization (Figure 1D). Furthermore, quantification of DAPI confirmed the removal of cell nuclei (Figure 1E), but the tubular structure of the liver was completely preserved. This indicates that the decellularization process effectively preserved the vascular and bile duct structures of the liver. This is extremely important for subsequent recellularization and functional recovery. [Examples]

[0030] Conceptual diagram of bioprint characterization of hydrogels using dECM

[0031] To utilize dECM as a hydrogel scaffold for tissue engineering, we performed further digestion using pepsin and acetic acid. However, the resulting solution had low viscosity and was unsuitable for use as a bio-link. To solve this problem, we considered adding GelMA, a hydrogel extracted from gelatin, known for its outstanding biocompatibility, primarily because it can support the adhesion and proliferation of various cell types. Furthermore, GelMA's properties can be easily adjusted to suit the mechanical properties of various tissues and provide appropriate variable viscosity, making it suitable for bioprinting processes. Therefore, we created a photocurable gel ink suitable for bioprinting by mixing dECM with GelMA (Figure 2A). 3D printing performed under microfluidic control showed that the bio-ink was smoothly extruded from the nozzle and rapidly solidified into a shape-retaining scaffold under UV irradiation (Figure 2B). When green fluorescent nanoparticles were added to the bioink, hydrogels with dECM were printed in 2, 4, and 6 layers, with the printed structure completely preserved, and the thickness of each layer was observed to increase (Figures 2C-D). This indicates that this Bio-link has good photocuring performance and the ability to form a multilayer scaffold. Scanning electron microscopy showed a large number of interconnected internal voids, indicating suitability for cell adhesion and proliferation (Figure 2E). Subsequently, the water absorption capacity of hydrogels with different concentrations of dECM was tested. When the hydrogels were immersed in phosphate-buffered saline (PBS), their total weight gradually increased over time, indicating that the scaffolds possessed hydrophilicity and water absorption capacity (Figure 2F). This indicates that the scaffolds can effectively absorb surrounding culture medium and nutrients in cell culture and tissue regeneration, providing a good fluid environment for cell proliferation. Subsequently, hydrogels with different concentrations of dECM were placed in PBS for 14 days to evaluate the stability of the scaffolds. As shown in Figure 2G, 3% dECM more effectively retained its structure and demonstrated sufficient stability for cell culture applications. [Examples]

[0032] Conceptual diagram for evaluating the biocompatibility of hydrogel scaffolds and maintaining cell function using dECM.

[0033] To evaluate the biocompatibility of dECM-based hydrogels, we selected iPSC-heps as the cell source and seeded cells on scaffolds printed with DelMA and 1.5% dECM hydrogel, and DelMA and 3% dECM hydrogel. Cell viability was assessed using viability staining on days 1, 3, 5, and 7 after seeding (Figure 3A). The results showed a significant increase in cell quantity and density over time, indicating that all scaffold types were suitable for cell adhesion and proliferation. Quantitative analysis of cells on day 7 revealed that the 3% dECM hydrogel had the highest cell viability, demonstrating its superior cell survival environment (Figure 3C). To further evaluate the function-sustaining effects of high-concentration dECM hydrogels, we examined albumin (ALB) secretion, CYP450 family expression, and mRNA levels of important hepatic transcription factors and functional genes in cells cultured on 1.5% and 3% dECM hydrogel scaffolds (Figure 3D). The results showed that a 3% dECM concentration significantly enhanced the expression of cellular function. Therefore, we selected a hydrogel based on 3% dECM and proceeded with subsequent experiments. To visually evaluate the adhesion of cells to the scaffold, we observed the scaffolds prepared with 3% dECM hydrogel using a scanning electron microscope (Figure 3B). As the magnification of the electron microscope gradually increased, the strong affinity of the cells to the scaffold surface became apparent. Examination of the cellular function expression status of the 3% dECM hydrogel scaffold using fluorescence staining revealed PCNA positivity, indicating that the cells possessed proliferative capacity. Furthermore, CYP3A4, ALB, and HNF4A staining confirmed the scaffold's ability to maintain albumin secretion, metabolic function, and physiological activity of hepatocytes (Figures 3E-F). Therefore, we concluded that a 3% dECM hydrogel-based hydrogel scaffold is suitable for application in in vivo liver regeneration. [Examples]

[0034] Conceptual diagram evaluating the therapeutic effect of ALF mice treated with dECM-based hydrogel scaffolding.

[0035] We induced acute liver failure (ALF) in mice by intraperitoneal injection of D-galactose (D-Gal), exposed the liver by making a 1 cm incision in the abdomen of the mice, and then orthotopically transplanted a hydrogel scaffold to evaluate its therapeutic effect on acute liver failure (Figures 4A, 4B). To study the colonization status of the hydrogel scaffold in the liver, it was stained with the cell membrane fluorescent dye 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DIR) before transplantation. In vivo imaging was performed on small animals after transplantation. As shown in Figure 4C, the hydrogel scaffold showed clearly strong fluorescence intensity in the liver region on day 7 after transplantation, and there was no migration to other parts of the body.

[0036] To further understand the therapeutic effect of hydrogel-mediated alanine-alanine fibrosis (ALF) using dECM, we examined indicators such as survival rate and liver function in each mouse group. These indicators included aspartate aminotransferase (AST) and alanine aminotransferase (ALT), both used to assess the degree of hepatocyte damage.

[0037] The experiment was set up with four groups, including the following:

[0038] Control group - normal mice

[0039] ALF group - Mice in which acute liver failure was induced.

[0040] Cell group - Mice in which acute liver failure was induced and treated with a simple iPSC-heps cell suspension.

[0041] Scaffold group - Mice in which acute liver failure was induced and treated with a microfluidically controlled 3D printed hydrogel using dECM prepared in Example 1 of the present invention.

[0042] In 7-day survival monitoring, the mortality rates in the Cell and Scaffold groups were lower than in the ALF group, and the survival rate was higher in the Scaffold group (Figure 4D). Furthermore, indicators of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) showed that the Scaffold group more effectively suppressed hepatocyte damage and promoted the recovery of liver function (Figures 4E, 4F). H&E staining of liver tissue showed extensive necrosis in the ALF group, but the necrotic area was significantly reduced in the two cell therapy groups, with the Scaffold group showing the smallest liver necrosis and the lowest Suzuki Score (Figures 4G, 4H). This indicates that recovery of liver damage was achieved after scaffold transplantation. We also evaluated the proliferative state of hepatocytes after transplantation using Ki-67 staining. Compared to the ALF group, both the Cell group and the Scaffold group showed significantly improved hepatocyte proliferation, with the Scaffold group exhibiting an even more pronounced effect on cell proliferation (Figures 5A, 5D). Similarly, TUNEL staining clearly showed a decrease in hepatocyte apoptosis levels in both the Cell and Scaffold groups, with the Scaffold group having a lower number of apoptotic cells (Figures 5B, 5E). Recognizing that inflammatory damage and oxidative stress responses are also major factors influencing liver regeneration, we stained liver tissue with CD68, HO-1, and Nrf2 to investigate the specific role of the Scaffold group in liver repair (Figures 5C, 5F-5H). The results showed higher CD68 expression in the Scaffold group compared to the ALF and Cell groups, indicating greater macrophage aggregation and greater resistance to inflammatory responses. Furthermore, activation of the Nrf2 / HO-1 antioxidant pathway in the Scaffold group also demonstrated that it can promote liver recovery by enhancing its antioxidant capacity. Therefore, we hypothesized that transplantation therapy in the scaffold group could repair the liver through its anti-inflammatory and antioxidant effects.

[0043] While preferred embodiments of the present invention have been described above, the present invention is not limited thereto. Within the spirit and scope of the present invention, any modifications, substitutions with equivalents, improvements, etc., made by those skilled in the art are included within the scope of protection of the present invention.

Claims

1. This microfluidically controlled 3D printed hydrogel is characterized by being a mixed material hydrogel scaffold fabricated using microfluidically controlled 3D printing technology, with a decellularized fish liver matrix combined with gelatin methacryloyl as the scaffold and human induced pluripotent stem cell-derived hepatocytes as the cell source.

2. 1) Production of hepatocytes derived from human induced pluripotent stem cells, 2) Preparation of a decellularized matrix from fish liver, 3) A microfluidically controlled 3D printed hydrogel using a decellularized fish liver matrix according to claim 1, characterized by a manufacturing procedure comprising: designing a 3D scaffold model; thoroughly mixing gelatin methacryloyl, a decellularized fish liver matrix prepared in step 2), human induced pluripotent stem cell-derived hepatocytes prepared in step (1), and a photoinitiator in water according to the proportions to form a bio-link; and solidifying it into a shape-retaining scaffold under ultraviolet irradiation.

3. The specific procedure in 2) above is: a. Prepare a fresh fish liver and complete blood vessels. b. Decellularization is performed by continuously perfusing fish liver with a surfactant solution at room temperature. c. Wash the material obtained in step b with phosphate-buffered saline. d. The 3D printed hydrogel according to claim 2, characterized in that the decellularized fish liver matrix obtained by the procedure c is dissolved in an aqueous acetic acid solution containing pepsin.

4. The 3D printed hydrogel according to claim 2, characterized in that the photoinitiator is 2-hydroxy-2-methyl-1phenyl-1propanone.

5. The 3D printed hydrogel according to claim 3, characterized in that the surfactant is sodium dodecyl sulfate or sodium deoxycholate.

6. The 3D printed hydrogel according to claim 3, characterized in that the concentration of the surfactant solution used in the above procedure b is 1%.

7. The 3D printed hydrogel according to claim 3, characterized in that the rate at which the fish liver is continuously perfused in step b is 10 mL / min and the time is 3 hours.

8. The 3D printed hydrogel according to claim 3, characterized in that the concentration of pepsin dissolved in the aqueous acetic acid solution containing pepsin in step d is 1%, and the concentration of acetic acid is 100 mM.

9. The 3D printed hydrogel according to claim 2, characterized in that the mass ratio in the reaction system of the decellularized fish liver matrix, gelatin methacryloyl, and photoinitiator is 1.5 to 3:7.5:

1.

10. Application of the 3D printed hydrogel according to any one of claims 1 to 9 in the preparation of a drug for liver regeneration.

Images