Microfluidic 3d-printed hydrogel based on fish liver decellularized extracellular matrix, and preparation method therefor and use thereof

Abstract

A microfluidic 3D-printed hydrogel based on fish liver decellularized extracellular matrix for liver regeneration and a preparation method. The hydrogel is prepared by means of combining fish liver decellularized extracellular matrix (dECM) and gelatin methacryloyl (GelMA), and loading induced pluripotent stem cell-derived hepatocytes (iPSC-heps) for liver regeneration. The microfluidic 3D-printed hydrogel based on fish liver decellularized extracellular matrix exhibits excellent biocompatibility, retains intact endogenous growth factors, maintains the biological activity of cells, ensures the effective encapsulation of the cells, and facilitates the robust functional expression of the iPSC-heps. After in-vivo transplantation, the survival rate and liver function of mice with acute liver failure are significantly improved, and liver regeneration and repair are promoted.

Description

Fish liver acellular matrix-based microfluidic 3D printing hydrogel and preparation method and application thereof TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical materials, and particularly relates to a fish liver acellular matrix-based microfluidic 3D printing hydrogel for liver regeneration and a preparation method thereof. BACKGROUND

[0002] Liver transplantation is an efficient method for treating end-stage liver disease, while the scarcity of donor livers limits its widespread clinical application. Tissue engineering is considered a promising method to replace donor livers due to its ability to construct functional liver tissue. Hydrogels are advanced materials that can be shaped into various structures required for liver transplantation, and have good biocompatibility and cell proliferation capacity for inducing pluripotent stem cell-derived hepatocytes (iPSC-heps). However, these hydrogels generally exhibit limited biological activity and are significantly different from the composition of natural liver tissue, and in addition, most hydrogels require complex manufacturing processes, causing potential biosafety problems and reducing therapeutic effects. Therefore, the need for innovative hydrogels with inherent biological activity and sufficient support for liver function has not been met.

[0003] As a natural material, acellular fish liver has excellent biocompatibility and retains intact endogenous growth factors, including collagen and glycosaminoglycans. Compared with terrestrial animal livers, fish livers from aquatic sources are abundant and inexpensive. In addition, microfluidic 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, and 3D printing allows personalized design of implantable hydrogels to meet specific clinical needs. With these advantages, the integration of microfluidics and 3D printing technology makes it possible to produce customizable liver regeneration hydrogels. Therefore, it is desirable to combine fish liver acellular hydrogels as a biological chain with microfluidic-assisted 3D printing technology to develop a new type of scaffold that effectively promotes liver repair. SUMMARY

[0004] The purpose of the present application is to solve the above technical problems, and we propose a simple microfluidic 3D printing technology to prepare a fish liver acellular matrix-based hydrogel required for liver regeneration for liver repair in situ. The hydrogel is prepared by a microfluidic 3D printing method, which is simple, versatile, and easy to mass-produce.

[0005] Technical solution: A kind of microfluidic 3D printing hydrogel based on fish liver acellular matrix, fish liver acellular matrix and gelatin methacryl are combined as a support, human induced pluripotent stem cell-derived hepatocytes are used as cell source, and the mixed material hydrogel support is prepared by microfluidic 3D printing technology.

[0006] The preparation steps of the microfluidic 3D printing hydrogel based on fish liver acellular matrix are as follows:

[0007] 1) Preparation of human induced pluripotent stem cell-derived hepatocytes;

[0008] 2) Preparation of fish liver acellular matrix;

[0009] 3) Design 3D support model, mix gelatin methacryl, fish liver acellular matrix prepared in step 2) and human induced pluripotent stem cell-derived hepatocytes prepared in step 1) and photoinitiator to form a biological link, and solidify into a support that maintains shape under ultraviolet irradiation.

[0010] The specific steps of step 2) are:

[0011] a. Take fresh fish liver and intact blood vessels;

[0012] b. Decellularize fish liver by continuously perfusing surfactant solution at room temperature;

[0013] c. Wash the material obtained in step b with phosphate buffered saline solution;

[0014] d. Dissolve the fish liver acellular matrix prepared in step c in an aqueous acetic acid solution containing pepsin.

[0015] Preferably, the photoinitiator is 2-hydroxy-2-methyl-1-phenyl-1-propanone.

[0016] Preferably, the surfactant is sodium dodecyl sulfate or sodium deoxycholate.

[0017] Preferably, the concentration of surfactant used in step b is 1% (g / 100mL, i.e. 1g of surfactant is dissolved in 100mL of water), the speed of continuously perfusing fish liver is 10 mL / min, and the time is 3 hours.

[0018] Preferably, the mass concentration of pepsin in the aqueous acetic acid 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.

[0019] Preferably, the mass ratio of fish liver acellular matrix, gelatin methacryl and photoinitiator in the reaction system is 1.5~3:7.5:1.

[0020] The application also provides application of the fish liver decellularized matrix-based microfluidic 3D printing hydrogel in preparation of a liver regeneration drug.

[0021] Advantages:

[0022] (1) The application designs a fish liver decellularized matrix-based microfluidic 3D printing hydrogel for liver regeneration, which has excellent biocompatibility.

[0023] (2) The fish liver decellularized matrix-based hydrogel for liver regeneration provided by the application is prepared by a microfluidic 3D printing technology, and has the advantages of simple method, convenient operation, strong repeatability, low technical requirement, strong universality, high flexibility and easy large-scale preparation.

[0024] (3) The fish liver decellularized matrix-based microfluidic 3D printing hydrogel for liver regeneration prepared by the application retains a unique fiber structure supporting cell adhesion and migration, maintains the biological activity of cells, guarantees effective cell encapsulation, and is conducive to robust functional expression of iPSC-hep. After in vivo transplantation, the survival rate and liver function of acute liver failure mice are significantly improved, and the regeneration and repair of the liver are promoted. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 shows the preparation and evaluation of fish liver decellularized matrix (dECM): Figure A shows the effect of 1% SDS on fish liver decellularization time, with 15, 30, 120, 240 and 360 minutes, respectively, and the scale is 0.5 cm; Figure B shows the DNA content in native and decellularized fish livers; Figure C shows the scanning electron microscope images, H&E and DAPI staining images of native and decellularized fish livers, with scales of 100 μm (i), 20 μm (ii, iii, iv) and 50 μm (v, vi), respectively; Figure D shows the immunofluorescence staining of laminin, collagen type I and collagen type IV in native and decellularized fish livers, with cell nuclei shown by DAPI staining, and the scale is 100 μm; Figure E shows DAPI quantitative fluorescence analysis of group D, showing the removal of cell nuclei after decellularization.

[0026] Figure 2 Schematic diagram of bio-printing characterization of dECM-based hydrogel: Figure A is the preparation of microfluidic 3D printing of dECM and GelMA mixed hydrogel; Figure B is a schematic diagram of the second, fourth and sixth layers of scaffolds using microfluidic 3D bio-printing; Figure C-D are fluorescence macrograph (C) and close-up (D) of the scaffolds after adding green fluorescent nanoparticles to the second, fourth and sixth layers; Figure E is a SEM image depicting the microstructure inside the bio-printed scaffold, the scale bars are 100 μm (i) and 50 μm (ii), respectively; Figure F-G are comparative analysis diagrams of the swelling rate (F) and degradation rate (G) of the bio-ink group without dECM (GelMA), 1.5% dECM bio-ink group and 3% dECM bio-ink group.

[0027] Figure 3 Schematic diagram of biocompatibility evaluation and cell function maintenance of dECM-based hydrogel scaffolds: Figure A is the seeding of iPSC-heps on GelMA, 1.5% dECM hydrogel and 3% dECM hydrogel printed scaffolds, and live-dead imaging at 1, 3, 5 and 7 days. The scale bar is 50 μm; Figure B is a scanning electron microscope showing the colonization of cells after 7 days of culture on dECM-based hydrogel scaffolds. The scale bars from top to bottom are 200 μm, 100 μm and 20 μm, respectively; Figure C is a quantitative analysis diagram of live-dead staining of cells cultured on three scaffolds for 7 days; Figure D is the mRNA expression level on 1.5% dECM-based hydrogel and 3% dECM-based hydrogel scaffolds; Figure E-F are immunofluorescence staining of cells on 3% dECM hydrogel scaffolds, the scale bar is 10 μm.

[0028] Figure 4 Schematic diagram of efficacy evaluation of dECM-based hydrogel scaffold transplantation for treatment of ALF mice: Figure A is a schematic diagram of ALF mouse model and hydrogel scaffold liver transplantation; Figure B is a schematic diagram of hydrogel scaffold adhering to the liver; Figure C is a small animal in vivo imaging showing the colonization of hydrogel scaffold on the liver; Figure D-F are schematic diagrams of the survival rate (D), aspartate aminotransferase (AST) (E) and glutamic-pyruvic transaminase (ALT) (F) levels of four groups of mice; Figure G-H are H&E staining (H) representing the degree of liver damage and the corresponding Suzuki score (G), the scale bar in (H) is 100 μm.

[0029] Figure 5 Schematic diagram of immunohistochemical evaluation of the effect of dECM-based hydrogel scaffold transplantation: Figures A-C are merged fluorescence images of representative Ki-67 (A), TUNEL (B) and CD68, Nrf2, HO-1 and DAPI (C) of liver samples of different groups; Figures D-H are fluorescence quantitative analysis diagrams of Ki-67 (D), TUNEL (E), CD68 (F), Nrf2 (G), HO-1 (H), the scale bar is 100 μm. DETAILED DESCRIPTION

[0030] In order to deepen the understanding of the present application, the present application will be further described below in conjunction with examples and drawings, which are only used to explain the present application and do not constitute a limitation on the protection scope of the present application.

[0031] The human induced pluripotent stem cells (hiPSC) used in the following examples were purchased from the Chinese Academy of Sciences Stem Cell Bank.

[0032] The concentration in the following examples means the mass of 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 each 100 mL of reaction solvent is 7.5 g.

[0033] Example 1 Preparation of fish liver acellular matrix-based microfluidic 3D printing hydrogel

[0034] (1) Preparation of human induced pluripotent stem cell-derived hepatocytes (hiPSC-hep) (The reference literature for the preparation steps is 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):

[0035] Human induced pluripotent stem cells (hiPSC) were cultured in 6-well plates (5x10 5Subsequently, the cells were cultured in RPMI1640 medium containing Activin a, BMP4, bFGF, B27 and Wnt3a for 1 day, and then transferred into RPMI1640 medium containing Activin a, BMP4 and bFGF for 3 days to stimulate the development of final endoderm cells. To promote the formation of hepatoblasts, the endoderm cells were cultured in RPMI1640 medium containing KGF, SB431542 and B27 for 2 days, and then in RPMI1640 medium containing KGF, BMP4, BMP2, bFGF and B27 for 3 days. To promote the differentiation of hepatocytes into hepatic progenitor cells (HPCs), the hepatoblasts were cultured in DMEM / F12 medium containing B27, forskolin, SB431542 EGF, CHIR99021, LPA, Dex and S1P for 6-8 days. To produce mature hepatocytes (hiPSC-Heps), the HPCs were cultured in Williams'E medium containing B27, forskolin and SB431542 for 21 days.

[0036] (2) Preparation of fish liver decellularized matrix: The intact liver and intact blood vessels were extracted from fresh fish. Then, the liver was continuously perfused with sodium dodecyl sulfate (SDS) at room temperature for decellularization. Subsequently, the liver was washed with phosphate buffered saline solution to eliminate any residual decellularizing agent. All the above operations were carried out 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.

[0037] (3) Preparation of microfluidic 3D printing hydrogel based on fish liver decellularized matrix

[0038] After sterilizing 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 mixed in water to form a biological link, which was immediately solidified into a shape-retaining hydrogel scaffold under ultraviolet irradiation. The concentration of dECM in the reaction system was 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.

[0039] Example 2 Evaluation of fish liver decellularized matrix (dECM)

[0040] Using 1% sodium dodecyl sulfate (SDS) as a decellularization agent, the decellularization process was facilitated by perfusion through the portal vein circulation of fish livers. As the perfusion time was prolonged, the overall color of the liver transitioned from opaque to transparent, progressing from the center to the periphery, with the intrahepatic ductal system and overall structural integrity being fully preserved (FIG. 1A). As shown in FIG. IB, DNA quantification analysis showed that there were less than 50 ng of double-stranded DNA per milligram of tissue, indicating that the decellularization process effectively washed out DNA from the fresh tissue. Compared with the original fish liver, scanning electron microscopy (SEM) showed that even after decellularization, the unique fibrous structure supporting cell adhesion and migration was still preserved (FIG. 1C). In addition, hematoxylin and eosin (H&E) staining and 4', 6-diamidino-2-phenylindole (DAPI) immunofluorescence staining showed that there was no obvious residual nucleus in the decellularized liver tissue, confirming that the cells in the liver were effectively removed. H&E staining showed that although the hepatocyte structure was dissolved, the intrinsic system of the liver remained intact, which meant that the underlying ECM and structural scaffolds essential for liver function were preserved, which was crucial 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 preserved after decellularization (FIG. ID). In addition, although DAPI quantification confirmed the elimination of cell nuclei (FIG. IE), the tubular structure of the liver remained intact, which indicated that the decellularization process effectively preserved the vascular and biliary structures of the liver, which was crucial for subsequent recellularization and functional recovery.

[0041] Example 3 Schematic diagram of bioprinting characterization based on dECM hydrogel

[0042] To utilize dECM as a hydrogel scaffold for tissue engineering, we performed further digestion using pepsin and acetic acid. However, the resulting solution viscosity was low, making it unsuitable as a bioink. To address this issue, we considered adding GelMA, a hydrogel extracted from gelatin, which is well known for its excellent biocompatibility, mainly because it can support the adhesion and proliferation of various cell types. In addition, the performance of GelMA can be well adjusted to match the mechanical properties of different tissues and provide appropriate adjustable viscosity, which is beneficial for the bio-printing process. Therefore, we mixed dECM with GelMA to manufacture a photocurable gel ink suitable for bio-printing (Figure 2A). 3D printing under microfluidic device operation showed that the bio-ink was smoothly extruded from the nozzle and rapidly solidified into a shape-retaining scaffold under UV irradiation (Figure 2B). By adding green fluorescent nanoparticles to the bio-ink, it can be seen that the dECM-based hydrogel was printed into 2, 4, and 6 layers, and the printed structure remained intact, with the thickness of each layer increasing (Figures 2C-D). This indicates that the bioink has good photocuring performance and the ability to form a multi-layered structure scaffold. Scanning electron microscopy shows a large number of interconnected internal pores, which are beneficial for cell adhesion and proliferation (Figure 2E). Subsequently, the water absorption capacity of dECM-based hydrogels of different concentrations was detected. When the hydrogel was immersed in phosphate buffered saline (PBS), its total weight gradually increased over time, indicating that the scaffold has hydrophilic and water absorption capacity (Figure 2F). This shows that the scaffold can effectively absorb the surrounding culture medium and nutrients in cell culture and tissue regeneration, providing a good fluid environment for cell proliferation. After that, dECM hydrogels of different concentrations were placed in PBS for 14 days to evaluate the stability of the scaffold. As shown in Figure 2G, 3% dECM more effectively maintained its structure, showing sufficient stability in cell culture applications.

[0043] Example 4 Biocompatibility evaluation and cell function maintenance of dECM-based hydrogel scaffold

[0044] To evaluate the biocompatibility of dECM hydrogel-based scaffolds, we chose iPSC-heps as the cell source and seeded them on DelMA and 1.5% dECM hydrogel, DelMA and 3% dECM hydrogel printed scaffolds, respectively. Cell viability was evaluated using live-dead staining at 1, 3, 5 and 7 days post-seeding (Figure 3A). 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-dead cells at 7 days showed that the cell survival rate was highest in 3% dECM hydrogel, indicating a more favorable cell survival environment (Figure 3C). To further evaluate the effect of high concentration dECM hydrogel 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 3D). The results showed that a 3% dECM concentration significantly enhanced the expression of cell function. Therefore, we chose 3% dECM-based hydrogel for subsequent experiments. To visually assess cell adhesion on the scaffold, we observed the 3% dECM hydrogel prepared scaffold under a scanning electron microscope (Figure 3B). As the magnification of the electron microscope gradually increased, it was clear that cells showed strong affinity to the scaffold surface. Fluorescent staining detected the expression of cell function on 3% dECM hydrogel scaffolds, and PCNA positivity indicated that cells had proliferative capacity, while CYP3A4, ALB, and HNF4A staining confirmed the ability of the scaffold to maintain liver cell albumin secretion, metabolic function, and biological activity (Figures 3E-F). Therefore, we believe that 3% dECM-based hydrogel scaffolds are suitable for in vivo liver regeneration applications.

[0045] Example 5 Schematic diagram of efficacy evaluation of dECM-based hydrogel scaffold transplantation in treatment of ALF mice

[0046] We induced acute liver failure (ALF) in mice by intraperitoneal injection of D-galactose (D-Gal), made a 1 cm incision on the upper abdomen of the mice to expose the liver, and then transplanted the hydrogel scaffold in situ to evaluate its efficacy in treating acute liver failure (Figures 4A, 4B). To study the colonization of the hydrogel scaffold in the liver, we stained it with fluorescent cell membrane dye 1,1'-octadecyl-3,3,3',3'-tetramethylindocyanine iodide (DIR) before transplantation. After transplantation, small animal in vivo imaging was performed, as shown in Figure 4C, the hydrogel scaffold showed a clear strong fluorescence intensity in the liver area 7 days after transplantation, and did not migrate to other parts of the body.

[0047] To further understand the efficacy of dECM hydrogel-based treatment of ALF, the survival rate and liver function of mice in each group were detected. The detection indexes include aspartate aminotransferase (AST) for evaluating the degree of liver cell damage, and alanine aminotransferase (ALT) for evaluating the degree of liver cell damage.

[0048] Four groups were set up in the experiment, including

[0049] Control group - normal mice;

[0050] ALF group - acute liver failure induced mice;

[0051] Cell group - acute liver failure induced mice, treated with simple iPSC-heps cell suspension;

[0052] Scaffold group - acute liver failure induced mice, treated with dECM-based microfluidic 3D printing hydrogel prepared in Example 1 of the application.

[0053] 7-day survival 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 4D). In addition, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) markers indicated that the Scaffold group more effectively controlled liver cell damage and promoted liver function recovery (Figures 4E, 4F). Liver tissue H&E staining showed that the liver tissue of the ALF group had extensive necrosis; however, in the two cell treatment groups, the necrotic area was significantly reduced, especially in the Scaffold group, which had the least liver necrosis, resulting in the lowest Suzuki score (Figures 4G, 4H). This indicates that liver damage is recovered after scaffold transplantation. In addition, we used Ki-67 staining to evaluate liver cell proliferation after cell transplantation. Compared with the ALF group, both the Cell and Scaffold groups significantly improved liver cell proliferation, with the Scaffold group having a more obvious effect on cell proliferation (Figures 5A, 5D). Similarly, TUNEL staining showed that the liver cell apoptosis level of the Cell and Scaffold groups was significantly reduced, and the Scaffold group had fewer apoptotic cells (Figures 5B, 5E). Given that inflammatory damage and oxidative stress response 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 (Figures 5C, 5F-H). The results showed that the expression of CD68 in the Scaffold group was higher compared with the ALF and Cell groups, indicating that macrophage aggregation was greater, and had resistance to inflammatory response. In addition, the activation of the Nrf2 / HO-1 antioxidant pathway in the Scaffold group also indicates that it can promote liver recovery by enhancing antioxidant capacity. Therefore, we believe that the transplantation treatment of the Scaffold group can promote liver repair through anti-inflammatory and antioxidant effects.

[0054] The above description is merely preferred embodiments of the application, and is not intended to limit the application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the application should be included in the protection scope of the application.

Claims

1. A microfluidic 3D printed hydrogel based on fish liver decellularized matrix, characterized in that, The application discloses a 3D printing hydrogel for liver regeneration.

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

3. The 3D printed hydrogel of claim 2, wherein, The specific steps of step 2) are as follows: a. taking fresh fish liver and complete blood vessels; b. continuously perfusing the fish liver with a surfactant solution at room temperature to perform decellularization; c. washing the material obtained in step b with a phosphate buffered saline solution; d. dissolving the fish liver acellular matrix prepared in step c in a pepsin-containing acetic acid aqueous solution.

4. The 3D printed hydrogel of claim 2, wherein, The photoinitiator is 2-hydroxy-2-methyl-1-phenyl-1-propanone.

5. The 3D printed hydrogel of claim 3, wherein, The surfactant is sodium dodecyl sulfate or sodium deoxycholate.

6. The 3D printed hydrogel of claim 3, wherein, The concentration of the surfactant solution used in step b is 1%.

7. The 3D printed hydrogel of claim 3, wherein, The speed of continuously perfusing the fish liver in step b is 10 mL / min, and the time is 3 hours.

8. The 3D printed hydrogel of claim 3, wherein, The concentration of pepsin in the pepsin-containing acetic acid aqueous solution in step d is 1%, and the concentration of acetic acid is 100 mM.

9. The 3D printed hydrogel of claim 2, wherein, The mass ratio of the fish liver acellular matrix, gelatin methacryl and photoinitiator in the reaction system is 1.5-3:7.5:

1.

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

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