Microfluidic 3D printing hydrogel based on fish liver acellular matrix and preparation method and application thereof

Abstract

The invention discloses a fish liver acellular matrix-based microfluidic 3D printing hydrogel for liver regeneration and a preparation method thereof, the fish liver acellular matrix (dECM) and gelatin methacryloyl (GelMA) are combined together, and induced pluripotent stem cell-derived hepatocytes (iPSC-hep) are loaded for liver regeneration. The microfluidic 3D printing hydrogel based on the fish liver acellular matrix has excellent biocompatibility, retains complete endogenous growth factors, retains the biological activity of cells, ensures effective cell encapsulation, and is beneficial to stable functional expression of iPSC-hep. After in-vivo transplantation, the survival rate and the liver function of acute hepatic failure mice are remarkably improved, and regeneration and repair of the liver are promoted.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, and in particular relates to a microfluidic 3D printing hydrogel based on fish liver decellularized matrix for liver regeneration and a preparation method thereof. Background Art

[0002] Liver transplantation is a highly effective method for treating end-stage liver disease, but the scarcity of donor livers limits its widespread clinical application. Tissue engineering, with its ability to construct functional liver tissue, is considered a promising alternative to liver donors. Hydrogels are advanced materials that can be molded into various structures required for liver transplantation and have good biocompatibility and cell proliferation capacity for induced pluripotent stem cell-derived hepatocytes (iPSC-heps). However, these hydrogels generally exhibit limited bioactivity and are significantly different from the composition of natural liver tissue. In addition, most hydrogels require complex manufacturing processes, causing potential biosafety issues and reducing therapeutic efficacy. Therefore, there is an unmet need for innovative hydrogels that have inherent bioactivity and provide sufficient support for liver function.

[0003] As a natural material, decellularized fish liver has excellent biocompatibility and retains complete endogenous growth factors, including collagen and glycosaminoglycans. Compared with terrestrial animal livers, fish livers from aquatic sources are abundant and low-cost. In addition, microfluidics technology is recognized for its precise control of fluid dynamics and is an advanced form of additive manufacturing for drug delivery, cell culture, and biosensors. 3D printing allows personalized design of implantable hydrogels to meet specific clinical needs. Taking advantage of these advantages, the integration of microfluidics and 3D printing technology makes it possible to produce customizable liver regeneration hydrogels. Therefore, it is highly desirable to combine fish liver decellularized hydrogels as a biochain with microfluidics-assisted 3D printing technology to develop a new scaffold that effectively promotes liver repair. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to solve the above-mentioned technical problems. We propose a simple microfluidic 3D printing technology to prepare a hydrogel based on fish liver decellularized matrix required for liver regeneration, which is used for in situ transplantation liver repair. The hydrogel is prepared by microfluidic 3D printing technology, which is simple, versatile and easy to mass produce.

[0005] Technical solution: A microfluidic 3D printed hydrogel based on fish liver decellularized matrix, which is a hybrid material hydrogel scaffold prepared by combining fish liver decellularized matrix and gelatin methacryloyl as a scaffold, using hepatocytes derived from human induced pluripotent stem cells as a cell source, and using microfluidic 3D printing technology.

[0006] The preparation steps of microfluidic 3D printed hydrogel based on fish liver decellularized matrix are as follows: 1) Preparation of human induced pluripotent stem cell-derived hepatocytes; 2) Preparation of fish liver decellularized matrix; 3) Designing a 3D scaffold model, fully mixing gelatin methacryloyl, the fish liver decellularized matrix prepared in step 2), the human induced pluripotent stem cell-derived hepatocytes prepared in step 1), and a photoinitiator to form a biological link, and solidifying it into a shape-retaining scaffold under ultraviolet irradiation.

[0007] The specific steps of step 2) are: a. Take fresh fish liver and intact blood vessels; b. Decellularization of fish liver by continuous perfusion with surfactant solution at room temperature; c. Washing the material obtained in step b with phosphate buffered saline; d. The fish liver decellularized matrix prepared in step c is dissolved in an acetic acid aqueous solution containing pepsin.

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

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

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

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

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

[0013] The present invention also provides the use of microfluidic 3D printing hydrogel based on fish liver decellularized matrix in the preparation of drugs for liver regeneration.

[0014] Beneficial effects:

[0015] (1) The present invention designs a microfluidic 3D printed hydrogel based on fish liver decellularized matrix for liver regeneration with excellent biocompatibility.

[0016] (2) The fish liver decellularized matrix-based hydrogel for liver regeneration provided by the present invention is prepared by a microfluidic 3D printing technology method, which is simple, easy to operate, highly repeatable, has low technical requirements, strong versatility, high flexibility, and is easy to prepare on a large scale.

[0017] (3) The microfluidic 3D printed hydrogel based on fish liver decellularized matrix for liver regeneration prepared by the present invention retains the unique fiber structure that supports cell adhesion and migration, maintains the biological activity of cells, ensures effective cell encapsulation, and is conducive to the robust functional expression of iPSC-hep. After in vivo transplantation, it significantly improved the survival rate and liver function of mice with acute liver failure and promoted liver regeneration and repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the preparation and evaluation of fish liver decellularized matrix (dECM): Figure A shows the effect of 1% SDS on the decellularization time of fish liver, which is 15, 30, 120, 240 and 360 minutes, respectively, and the scale bar is 0.5 cm; Figure B shows the DNA content in native and decellularized fish liver; Figure C shows the scanning electron microscopy, H&E and DAPI staining images of native and decellularized fish liver. The scale bars are 100 μm (i), 20 μm (ii, iii, iv) and 50 μm (v, vi), respectively; Figure D shows the immunofluorescence staining of laminin, type I collagen and type IV collagen in native and decellularized fish liver, and the cell nucleus is shown by DAPI staining, and the scale bar is 100 μm; Figure E shows the quantitative fluorescence analysis of DAPI in group D, showing the removal of cell nuclei after decellularization.

[0019] Figure 2 Schematic diagram of bioprinting characterization based on dECM hydrogel: Figure A shows the preparation of microfluidic 3D printed dECM and GelMA mixed hydrogel; Figure B shows a schematic diagram of the 2nd, 4th and 6th layer scaffolds using microfluidic 3D bioprinting; Figures CD show the fluorescent macroscopic image (C) and close-up image (D) of the scaffolds after adding green fluorescent nanoparticles to the 2nd, 4th and 6th layers; Figure E shows the SEM image depicting the microstructure inside the bioprinted scaffold, with scales of 100 μm (i) and 50 μm (ii), respectively; Figures FG show comparative analysis of the swelling rate (F) and degradation rate (G) of the bio-ink group without dECM (GelMA), the 1.5% dECM bio-ink group and the 3% dECM bio-ink group.

[0020] Figure 3Schematic diagram of biocompatibility evaluation and cell function maintenance of dECM-based hydrogel scaffolds: Figure A shows iPSC-heps seeded on GelMA, 1.5% dECM hydrogel, and 3% dECM hydrogel printed scaffolds, and live-death imaging was performed at 1, 3, 5, and 7 days. The scale bar is 50 μm. Figure B is a schematic diagram of cell colonization after 7 days of culture on dECM-based hydrogel scaffolds by scanning electron microscopy. The scale bars are 200 μm, 100 μm, and 20 μm from top to bottom, respectively; Figure C is a quantitative analysis of live-death staining after 7 days of cell culture on the three scaffolds; Figure D is the mRNA expression level on 1.5% dECM-based hydrogel and 3% dECM-based hydrogel scaffolds; Figure EF is immunofluorescence staining of cells on 3% dECM hydrogel scaffolds, and the scale bar is 10 μm.

[0021] Figure 4 Schematic diagram of the efficacy evaluation of dECM-based hydrogel scaffold transplantation in the treatment of ALF mice: Figure A is a schematic diagram of the ALF mouse model and hydrogel scaffold liver transplantation; Figure B is a schematic diagram of the hydrogel scaffold adhering to the liver; Figure C is a schematic diagram of the in vivo imaging of small animals showing the colonization of the hydrogel scaffold in the liver; Figures DF are schematic diagrams of the survival rate (D), aspartate aminotransferase (AST) (E), and alanine aminotransferase (ALT) (F) levels of the four groups of mice; Figures GH are H&E staining (H), indicating the degree of liver damage and the corresponding Suzuki score (G), and the scale bar in (H) is 100 μm.

[0022] Figure 5 Schematic diagram of immunohistochemical evaluation of the therapeutic effect of dECM-based hydrogel scaffold transplantation: Figures AC are representative merged fluorescence images of Ki-67 (A), TUNEL (B), and CD68, Nrf2, HO-1, and DAPI (C) of liver samples from different groups; Figures DH are fluorescence quantitative analysis images of Ki-67 (D), TUNEL (E), CD68 (F), Nrf2 (G), and HO-1 (H), with a scale bar of 100 μm. DETAILED DESCRIPTION

[0023] In order to deepen the understanding of the present invention, the present invention will be further described in detail below in conjunction with embodiments and drawings. The embodiments are only used to explain the present invention and do not constitute a limitation on the protection scope of the present invention.

[0024] The human induced pluripotent stem cells (hiPSCs) used in the following examples were purchased from the Stem Cell Bank of the Chinese Academy of Sciences.

[0025] In the following examples, the concentration refers to the mass of the solute in 100 mL of solvent. For example, the concentration of GelMA in the reaction system is 7.5%, which means that the mass of GelMA in every 100 mL of reaction solvent is 7.5 g.

[0026] Example 1 Preparation of microfluidic 3D printed hydrogel based on fish liver decellularized matrix Preparation of human induced pluripotent stem cell-derived hepatocytes (hiPSC-hep) (the preparation steps are cited in the prior art document Chen, Sitong et al. Hepatic spheroids derived from human induced pluripotentstem cells in bio-artificial liver rescue porcine acute liver failure. Cell Research, (2019) 0: 1-3): Human induced pluripotent stem cells (hiPSCs) were cultured in mTeSR1 medium containing Matrigel at 37°C and 5% CO2 in 6-well plates (5 × 10 cells per well). 5 cells). Subsequently, the cells were cultured in RPMI1640 medium containing activin a, BMP4, bFGF, B27 and Wnt3a for 1 day, and then transferred to RPMI1640 medium containing activin a, BMP4 and bFGF for 3 days to stimulate the final endoderm cell development. To promote the formation of hepatoblasts, the endoderm cells were cultured in RPMI1640 medium containing KGF, SB431542 and B27 for 2 days, and then cultured in RPMI1640 medium containing KGF, BMP4, BMP2, bFGF and B27 for 3 days. In order 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 in Williams' E medium containing B27, forskolin, and SB431542 for 21 days.

[0027] (2) Preparation of fish liver decellularized matrix: The intact liver and intact blood vessels were extracted from fresh fish. Then, the liver was decellularized by continuous perfusion with sodium dodecyl sulfate (SDS) at room temperature. Subsequently, the liver was washed with phosphate-buffered saline to eliminate any residual decellularizing agent. All the above operations were performed under sterile conditions. The prepared fish liver decellularized matrix (dECM) was dissolved with 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.

[0028] (3) Preparation of microfluidic 3D printed hydrogel based on fish liver decellularized matrix After disinfecting the microfluidic printing device, a 3D scaffold model was designed for printing. The fish liver decellularized matrix prepared in step (2), GelMA, hiPSC-heps prepared in step (1), and photoinitiator HMPP (2-hydroxy-2-methyl-1-phenyl-1-propanone) were fully mixed in water to form a biological link, which was immediately solidified into a shape-retaining hydrogel scaffold under ultraviolet irradiation. The concentrations of the 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 was 10 7 cells.

[0029] Example 2 Evaluation of fish liver decellularized matrix (dECM) 1% sodium dodecyl sulfate (SDS) was used as a decellularizing agent and circulated through the portal vein of the fish liver to promote the decellularization process. As the perfusion time increased, the overall color of the liver changed from opaque to transparent, advancing from the center to the periphery, and the intrahepatic ductal system and overall structural integrity were fully preserved ( Figure 1 A). Figure 1 As shown in B, DNA quantification analysis showed less than 50 ng of double-stranded DNA per mg of tissue, indicating that the decellularization process effectively eluted DNA from fresh tissue. Compared with the original fish liver, scanning electron microscopy (SEM) showed that the unique fiber structure supporting cell adhesion and migration was retained even after decellularization ( Figure 1C). In addition, hematoxylin and eosin (H&E) staining and 4′,6-diamidino-2-phenylindole (DAPI) immunofluorescence staining showed that there were no obvious residual nuclei in the decellularized liver tissue, confirming the effective removal of cells in the liver. H&E staining can be observed that although the liver cell structure is dissolved, the intrinsic system of the liver remains intact, which means that the basic ECM and structural scaffolds that are essential for liver function are retained, which is essential for maintaining the structure and function of the organ. On this basis, we stained the key ECM components of tissue regeneration, and the results showed that components such as laminin, type I collagen, and type IV collagen were retained after decellularization ( Figure 1 D). In addition, although DAPI quantification confirmed the elimination of nuclei ( Figure 1 E), but the tubular structure of the liver remained intact, indicating that the decellularization process effectively preserved the vascular and biliary structures of the liver, which are critical for subsequent recellularization and functional recovery.

[0030] Example 3 Schematic diagram of bioprinting characterization based on dECM hydrogel To utilize dECM as a hydrogel scaffold for tissue engineering, we performed further digestion using pepsin and acetic acid. However, the resulting solution had a low viscosity, making it unsuitable as a bioconnector. To address this issue, we considered adding GelMA, a hydrogel derived from gelatin that is known for its excellent biocompatibility, primarily due to its ability to support the adhesion and proliferation of various cell types. Furthermore, the properties of GelMA can be well tuned to match the mechanical properties of different tissues and provide an appropriately tunable viscosity that facilitates the bioprinting process. Therefore, we mixed dECM with GelMA to create a photocurable gel ink suitable for bioprinting ( Figure 2 A). 3D printing under the operation of a microfluidic device shows that the bio-ink is smoothly extruded from the nozzle and quickly solidified into a shape-retaining scaffold under ultraviolet light ( Figure 2 B). When green fluorescent nanoparticles are added to the bio-ink, it can be seen that the dECM-based hydrogel is printed into 2, 4, and 6 layers. The printed structure remains intact and the thickness of each layer increases ( Figure 2 CD). This indicates that the biochain has good photocuring properties and the ability to form multilayered structural scaffolds. Scanning electron microscopy shows a large number of interconnected internal pores, which are conducive to cell adhesion and proliferation ( Figure 2 E). Subsequently, the water absorption capacity of dECM-based hydrogels with different concentrations was tested. The total weight of the hydrogels gradually increased over time after being immersed in phosphate-buffered saline (PBS), indicating that the scaffolds were hydrophilic and capable of water absorption ( Figure 2F). This indicates that the scaffold can effectively absorb the surrounding culture medium and nutrients during cell culture and tissue regeneration, providing a good fluid environment for cell proliferation. Afterwards, dECM hydrogels with different concentrations were placed in PBS for 14 days to evaluate the stability of the scaffold. Figure 2 As shown in G, 3% dECM maintained its structure more effectively and showed sufficient stability for cell culture applications.

[0031] Example 4 Schematic diagram of biocompatibility evaluation and cell function maintenance of dECM-based hydrogel scaffolds To evaluate the biocompatibility of dECM-based hydrogels, we selected iPSC-hepsin as the cell source and seeded them on scaffolds printed with DelMA and 1.5% dECM hydrogels and DelMA and 3% dECM hydrogels, respectively. Cell viability was assessed using live-dead staining at 1, 3, 5, and 7 days after seeding ( Figure 3 A). The results showed that the number and density of cells increased significantly over time, indicating that all scaffold types were conducive to cell adhesion and growth. Quantitative analysis of live and dead cells on day 7 revealed that the 3% dECM hydrogel had the highest cell survival rate, indicating a more favorable cell survival environment ( Figure 3 C). To further evaluate the effect of high-concentration dECM hydrogels on maintaining cell function, we detected albumin (ALB) secretion, CYP450 family expression, and mRNA levels of key liver transcription factors and functional genes in cells cultured on 1.5% and 3% dECM hydrogel scaffolds ( Figure 3 D). The results showed that 3% dECM concentration can significantly enhance the expression of cell function. Therefore, we chose 3% dECM-based hydrogel for subsequent experiments. In order to intuitively evaluate the adhesion of cells on the scaffold, we performed scanning electron microscopy observations on the scaffold prepared with 3% dECM hydrogel ( Figure 3 B). As the magnification of the electron microscope gradually increased, it was clear that the cells showed a strong affinity for the scaffold surface. Fluorescence staining was used to detect the expression of cell function in the 3% dECM hydrogel scaffold. PCNA positivity indicated that the cells had the ability to proliferate, while CYP3A4, ALB and HNF4A staining confirmed the ability of the scaffold to maintain hepatocyte albumin secretion, metabolic function and biological activity ( Figure 3 EF). Therefore, we believe that 3% dECM-based hydrogel scaffolds are suitable for in vivo liver regeneration applications.

[0032] Example 5 Schematic diagram of the efficacy evaluation of dECM-based hydrogel scaffold transplantation in the treatment of ALF mice We induced acute liver failure (ALF) in mice by intraperitoneal injection of D-galactose (D-Gal), made a 1 cm incision in the upper abdomen of the mice to expose the liver, and then orthotopically transplanted the hydrogel scaffold to evaluate its efficacy in treating acute liver failure ( Figure 4 A, 4B). To investigate the colonization of the hydrogel scaffold in the liver, it was stained with the fluorescent cell membrane dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindolylcarbocyanine iodide (DIR) before transplantation. After transplantation, in vivo imaging of small animals was performed, such as Figure 4 As shown in C, the hydrogel scaffold showed obvious strong fluorescence intensity in the liver area 7 days after transplantation and did not migrate to other parts of the body.

[0033] In order to further understand the efficacy of dECM hydrogel-based treatment of ALF, the survival rate of mice in each group, liver function and other indicators were tested. The test indicators included aspartate aminotransferase (AST) to evaluate the degree of liver cell damage and alanine aminotransferase (ALT) to evaluate the degree of liver cell damage.

[0034] The experiment set up four groups, including Control group—normal mice; ALF group—mice induced with acute liver failure; Cell group: mice induced with acute liver failure, treated with simple iPSC-heps cell suspension; Scaffold group: mice induced with acute liver failure, treated with the dECM-based microfluidic 3D printed hydrogel prepared in Example 1 of the present invention.

[0035] The 7-day survival rate monitoring showed that the mortality rate of the Cell and Scaffold groups was lower than that of the ALF group, and the survival rate of the Scaffold group was higher ( Figure 4 D). In addition, the alanine aminotransferase (ALT) and aspartate aminotransferase (AST) markers showed that the Scaffold group more effectively controlled liver cell damage and promoted liver function recovery ( Figure 4 E, 4F). H&E staining of liver tissue showed extensive necrosis in the ALF group; however, the necrotic area was significantly reduced in both cell-treated groups, especially in the Scaffold group, which had minimal liver necrosis, resulting in the lowest Suzuki score ( Figure 4 G, 4H). This indicates that liver damage was restored after scaffold transplantation. In addition, we used Ki-67 staining to evaluate the proliferation of hepatocytes after transplantation. Compared with the ALF group, both the Cell group and the Scaffold group significantly improved hepatocyte proliferation, and the effect of the Scaffold group on cell proliferation was more obvious ( Figure 5A, 5D). Similarly, TUNEL staining showed that the level of hepatocyte apoptosis in the Cell group and the Scaffold group was significantly reduced, and the number of apoptotic cells in the Scaffold group was even smaller ( Figure 5 B, 5E). Given that inflammatory damage and oxidative stress are also major factors affecting liver regeneration, in order to explore the specific role of the scaffold group in liver repair, we performed CD68, HO-1, and Nrf2 staining on liver tissue ( Figure 5 C, 5F-H). The results showed that the expression of CD68 was higher in the Scaffold group compared with the ALF group and the Cell group, indicating that macrophages were more aggregated and resistant to inflammatory responses. In addition, the activation of the Nrf2 / HO-1 antioxidant pathway in the Scaffold group also indicated that it could promote liver recovery by enhancing antioxidant capacity. Therefore, we believe that transplantation therapy in the Scaffold group may promote liver repair through anti-inflammatory and antioxidant effects.

[0036] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A microfluidic 3D printing hydrogel based on fish liver decellularized matrix, characterized in that: It is a hybrid material hydrogel scaffold prepared by combining fish liver decellularized matrix and gelatin methacryloyl as a scaffold, using human induced pluripotent stem cell-derived hepatocytes as a cell source, and using microfluidic 3D printing technology.

2. The microfluidic 3D printing hydrogel based on fish liver decellularized matrix according to claim 1, characterized in that: The preparation steps are as follows: 1) Preparation of human induced pluripotent stem cell-derived hepatocytes; 2) Preparation of fish liver decellularized matrix; 3) Designing a 3D scaffold model, mixing gelatin methacryloyl, the fish liver decellularized matrix prepared in step 2), the human induced pluripotent stem cell-derived hepatocytes prepared in step 1), and a photoinitiator in proportion in water to form a biological link, and solidifying the mixture into a shape-retaining scaffold under ultraviolet irradiation.

3. The 3D printed hydrogel according to claim 2, characterized in that: The specific steps of step 2) are: a. Take fresh fish liver and intact blood vessels; b. Decellularization of fish liver by continuous perfusion with surfactant solution at room temperature; c. Washing the material obtained in step b with phosphate buffered saline; d. The fish liver decellularized matrix prepared in step c is dissolved in an acetic acid aqueous solution containing pepsin.

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

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 step b is 1%.

7. The 3D printed hydrogel according to claim 3, characterized in that: In step b, the fish liver is continuously perfused at a rate of 10 mL / min for 3 hours.

8. The 3D printed hydrogel according to claim 3, characterized in that: In step d, the concentration of pepsin in the acetic acid aqueous solution of pepsin 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 of the fish liver decellularized matrix, gelatin methacryloyl and photoinitiator in the reaction system is 1.5-3:7.5:

1.

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

Images