Construction method of in-situ biological 3D printing artificial liver patch for treating hepatic failure

Abstract

The invention belongs to the field of biomedicine, and particularly relates to a construction method of an in-situ biological 3D printing artificial liver patch for treating hepatic failure. The main component of the artificial liver patch disclosed by the invention is composite hydrogel, and the composite hydrogel comprises hyaluronic acid (HA), gelatin (Gelatin), sodium alginate (sodium alginate) and methacrylated gelatin (GelMA). The composite hydrogel has good 3D printing performance, large-scale construction of the artificial liver patch can be achieved through biological 3D printing, and a potential solution is provided for transplantable biological artificial liver tissue. The artificial liver patch adopts an extrusion type printing mode, the forming speed is high, internal liver cells can still keep high activity, reconstruction of liver basic functions (glycogen storage, drug metabolism, secretory protein synthesis and the like) can be guaranteed, and the artificial liver patch has important potential in treatment of liver failure diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a method for constructing an in situ biological 3D-printed artificial liver patch for treating liver failure. Background Art

[0002] As the body's primary metabolic and detoxification organ, the liver is susceptible to viral infections (such as HBV and HCV), alcohol abuse, non-alcoholic fatty liver disease (NAFLD), drug toxicity, and inherited metabolic disorders, leading to serious conditions such as hepatitis, liver fibrosis, cirrhosis, and even liver cancer. Liver transplantation is currently an effective treatment for end-stage liver disease, but donor shortages, immune rejection, and surgical risks hinder its widespread use.

[0003] Currently, tissue engineering technology has been used to construct liver tissue models, but traditional methods have the following problems: two-dimensional culture systems cannot simulate the three-dimensional microenvironment in the body, resulting in decreased liver cell function and difficulty in maintaining long-term culture; although hydrogel scaffolds can provide partial three-dimensional support, they have a single structure and lack vascularization, which limits cell survival and functionality; traditional liver tissue construction methods still have room for optimization in terms of cell arrangement, spatial organization and functional maintenance, making it difficult to meet the needs of clinical transplantation or in vitro research. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for preparing an in situ biological 3D printed artificial liver patch that can be used to treat liver diseases.

[0005] It's important to note that 3D bioprinting is a recently emerging in vitro three-dimensional culture method that precisely constructs tissue structures through layer-by-layer printing. Compared to traditional tissue engineering methods, 3D bioprinting can precisely control the distribution of cells in three dimensions, making them more similar to in vivo tissue structures. Furthermore, bio-inks can be used to regulate material composition, cell types, and growth factor distribution, optimizing the tissue microenvironment. In clinical applications, it can be used to customize the printing of liver tissue fragments tailored to the patient's condition and anatomical structure, improving treatment efficacy.

[0006] Bio-3D printed in situ liver patch transplantation enhances the integration efficiency of in vitro expanded and cultured hepatocytes into the liver. The patch can naturally receive blood supply from the hepatic artery and portal vein, enabling hepatocytes to carry out metabolic activities efficiently. It is expected to become a new model for the treatment of liver disease and provide patients with end-stage liver disease with a better alternative than traditional liver transplantation.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention provides a method for constructing an in situ biological 3D printed artificial liver patch for treating liver failure, comprising the following steps:

[0009] S1, hyaluronic acid, gelatin, sodium alginate and methacrylated gelatin were weighed, placed in phosphate buffered saline (PBS) solution, and stirred thoroughly at 60°C to prepare a composite hydrogel;

[0010] S2: At 37°C, the composite hydrogel was incubated with mouse primary liver cells (final cell concentration was 5×10 6 / mL) were mixed in a volume ratio of 1:1 to prepare the bio-ink required for printing and refrigerated;

[0011] S3, loading the bio-ink into a sterile syringe and printing using a 3D bio-printer to obtain a liver print;

[0012] S4, the liver print is placed in a CaCl2 solution for rapid cross-linking, and then cultured for a long time to obtain an in situ biological 3D printed artificial liver patch for the treatment of liver failure.

[0013] Optionally, in step S1, based on the liver components, the mass ratio of hyaluronic acid, gelatin, sodium alginate and methacrylated gelatin is selected to be 0.75:7.5:3:7.5 to simulate the liver extracellular matrix (ECM) components, optimize the cell microenvironment, and promote the expression of liver cell function and post-transplantation integration.

[0014] Optionally, in step S2, the prepared hydrogel containing liver cells is refrigerated at 4°C for 10-30 minutes.

[0015] Optionally, in step S3, select a suitable extrusion nozzle diameter, adjust the printing nozzle temperature to 10-15°C, adjust the printing platform temperature to 5-10°C, and use a 2-4 mm 3 The cell-containing bio-ink was printed in a 6 cm sterile culture dish at an extrusion speed of / s.

[0016] Optionally, in step S4, the mass concentration of the CaCl2 solution is 3%.

[0017] Furthermore, in step S4, the cells are cultured in a 37° C., 5% CO 2 incubator using DMEM / F-12 medium containing acetylcysteine, nicotinamide, recombinant human growth factor, Wnt3a protein, 5% fetal bovine serum and double antibody, and the medium is changed regularly to maintain activity.

[0018] It should be noted that the in situ biological 3D printed artificial liver patch constructed by the present invention can be used for the treatment of liver diseases. This artificial liver patch simulates the physiological structure and function of the liver through three-dimensional culture technology, and can perform liver metabolism, detoxification and other functions. The patch can use the host vascular network to improve the survival rate of transplanted tissue and reduce the risk of hypoxic necrosis. It is suitable for different degrees and types of liver damage and can be used for local repair or overall liver regeneration.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1) The artificial liver patch of this invention is primarily composed of a composite hydrogel, including hyaluronic acid (HA), gelatin, sodium alginate, and gelatin methacryloyl (GelMA). This composite hydrogel exhibits excellent 3D printing properties, enabling the scalable construction of artificial liver patches through bio-3D printing, providing a potential solution for the development of transplantable bioartificial liver tissue.

[0021] 2) The artificial liver patch described in the present invention adopts an extrusion printing mode, which has a fast molding speed, and the liver cells inside can still maintain a high level of activity, which can ensure the reconstruction of basic liver functions (glycogen storage, drug metabolism, and synthesis of secretory proteins, etc.), and has important potential in the treatment of liver failure.

[0022] 3) The artificial liver patch described in this invention is highly biomimetic, simulating the liver's tissue structure and function to provide a more realistic transplant material. It can be mass-produced in vitro using 3D bioprinting technology, potentially addressing the shortage of liver donors in clinical practice. The artificial liver patch achieves centimeter-scale tissue construction and demonstrates significant advantages in liver function expression. This patch is not only useful for in vitro liver disease research and drug screening, but also offers new treatment options for patients with end-stage liver disease, promising broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0024] Figure 1 The Young's modulus diagram of the composite hydrogel and other commonly used hydrogels;

[0025] Figure 2 3D-printed liver patches for in vitro biological culture;

[0026] Figure 3 Microscope photos of the 3D bioprinted liver patch cultured in vitro for 1, 5, 10, and 14 days;

[0027] Figure 4 Cell viability of the 3D bioprinted liver patch cultured in vitro for 1, 4, and 7 days;

[0028] Figure 5 To enable the bio-3D printed liver patch to perform basic liver functions in vitro;

[0029] Figure 6 The integration of the 3D bioprinted liver patch after in vivo transplantation. DETAILED DESCRIPTION

[0030] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0031] The term "embodiment" is used herein specifically to describe any embodiment as "exemplary," and should not be construed as superior or preferable to other embodiments. Performance indicators in the embodiments of this application were tested using conventional testing methods in the art, unless otherwise specified. It should be understood that the terms used in this application are intended solely to describe specific implementations and are not intended to limit the disclosure herein.

[0032] Unless otherwise specified, the technical and scientific terms used herein have the same meanings as commonly understood by ordinary technicians in the technical field to which this application belongs; other experimental methods and technical means not specifically specified in this application refer to experimental methods and technical means commonly used by ordinary technicians in this field.

[0033] In order to better illustrate the content of this application, numerous specific details are provided in the specific examples below. It should be understood by those skilled in the art that this application can be implemented without certain specific details. In the examples, some methods, means, instruments, equipment, etc. well known to those skilled in the art are not described in detail in order to highlight the main purpose of this application.

[0034] Under the premise of no conflict, the technical features disclosed in the embodiments of this application can be combined arbitrarily, and the resulting technical solutions belong to the contents disclosed in the embodiments of this application.

[0035] The present invention discloses a method for constructing an in situ biological 3D-printed artificial liver patch for treating liver failure.

[0036] For a better understanding of the present invention, the present invention is further specifically described below through the following examples, but it should not be understood as limiting the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above invention content are also considered to fall within the scope of protection of the present invention.

[0037] Example 1

[0038] A method for constructing an in situ biological 3D printed artificial liver patch for treating liver failure comprises the following steps:

[0039] 1. A composite hydrogel, based on the main components of the liver extracellular matrix, simulates the liver cell microenvironment and promotes the proliferation and maturation of liver cells, which is characterized by comprising hyaluronic acid, gelatin, sodium alginate and methacrylated gelatin.

[0040] The mass concentration of hyaluronic acid is 0.75%; the mass concentration of gelatin is 7.5%; the mass concentration of sodium alginate is 3%; and the mass concentration of methacrylated gelatin is 7.5%.

[0041] 2. An extrusion 3D printing method: The composite hydrogel was mixed with mouse primary liver cells (final concentration of 5×10 6 / mL) to prepare the bio-ink required for printing. The prepared hydrogel containing liver cells was placed in a refrigerator at 4°C for 18 minutes. Select an appropriate extrusion nozzle diameter, adjust the printing nozzle temperature to 15°C, and the printing platform temperature to 10°C. Use a 3mm 3 The cell-containing bio-ink was printed in a 6cm sterile culture dish at an extrusion speed of 1 / s. The printed liver was then placed in a 3% CaCl2 solution for rapid crosslinking. The printed liver was then incubated in a 37°C, 5% CO2 incubator using DMEM / F-12 culture medium containing acetylcysteine, nicotinamide, recombinant human growth factor, Wnt3a protein, 5% fetal bovine serum, and double-stranded antibodies for long-term culture, yielding the in situ bio-3D-printed artificial liver patch for treating liver failure.

[0042] To further demonstrate the beneficial effects of the present invention and to provide a better understanding of the present invention, the following test examples are provided to further illustrate the technical features disclosed in the present invention, but they are not to be construed as limiting the present invention. Other improvements made by those skilled in the art based on the above-mentioned invention without inventive steps are also deemed to fall within the scope of protection of the present invention.

[0043] Based on the main components of the liver extracellular matrix, the liver cell microenvironment was simulated, the concentration and component ratio of the hydrogel were optimized, the Young's modulus was measured, and the Young's modulus results of each hydrogel were compared to screen out a composite hydrogel (see Figure 1), the composite hydrogel (Composite hydrogel) is the hydrogel prepared in Example 1 above, which can promote the proliferation and maturation of liver cells. The liver cells were separated from the mouse liver, and the proliferation was induced using the hepatocyte expansion medium (HEM). After 4 days, the cells were digested with 2.5% trypsin and mixed with the composite hydrogel (the mass concentration of hyaluronic acid was 0.75%; the mass concentration of gelatin was 7.5%; the mass concentration of sodium alginate was 3%; the mass concentration of methacryloyl gelatin was 7.5%) in a ratio of 1:1. The mixture was refrigerated at 4°C for 20 minutes, and its printing performance was measured. Then, a suitable extrusion nozzle diameter was selected, the printing nozzle temperature was adjusted to 10-15°C, and the printing platform temperature was adjusted to 5-10°C. Use 2-4mm 3 The cell-containing bio-ink was printed in a 6 cm sterile culture dish at an extrusion speed of / s. The printed liver was placed in a 3% CaCl2 solution for rapid cross-linking and then cultured in vitro for a long time. After culturing for 3 days, its morphology was observed under a microscope. Figure 2 .

[0044] The printed artificial liver patch was cultured in vitro for 1, 5, 10, and 14 days and then imaged under a microscope. Figure 3 .

[0045] The printed artificial liver patch was cultured in vitro for 1, 4, and 7 days, and then subjected to live-dead staining and bright field observation. The patch was imaged under a microscope, and the obtained staining results were integrated. Figure 4 (BF: bright field, Live: live cell staining, Dead: dead cell staining, Merge: integrated staining results).

[0046] The liver function of the printed artificial liver cultured in vitro was evaluated at 7 days, showing an increase in the expression levels of liver function genes (including Alb, Ttr, and Cyp3a11), indicating further maturation of cells within the artificial liver patch. Figure 5 .

[0047] The printed 3D bioprinted liver patch was transplanted into mice, and it was observed that the artificial liver patch was well integrated with the mouse liver. Figure 6 .

[0048] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for constructing an in situ biological 3D printed artificial liver patch for treating liver failure, characterized in that: The steps include: S1, hyaluronic acid, gelatin, sodium alginate and methacrylated gelatin were weighed, placed in phosphate buffered saline (PBS) solution, and stirred thoroughly at 60°C to prepare a composite hydrogel; S2, mixing the composite hydrogel with mouse primary liver cells at 37°C to prepare the bio-ink required for printing and refrigerating; S3, loading the bio-ink into a sterile syringe and printing using a 3D bio-printer to obtain a liver print; S4, the liver print is placed in a CaCl2 solution for rapid cross-linking, and then cultured for a long time to obtain an in situ biological 3D printed artificial liver patch for the treatment of liver failure.

2. The method for constructing an in situ biological 3D printed artificial liver patch for treating liver failure according to claim 1, characterized in that: In step S1, the mass ratio of the hyaluronic acid, gelatin, sodium alginate and methacrylated gelatin is 0.75:7.5:3:7.

5.

3. The method for constructing an in situ biological 3D printed artificial liver patch for treating liver failure according to claim 1, characterized in that: It is characterized by: In step S2, the prepared hydrogel containing liver cells is refrigerated at 4° C. for 10-30 minutes.

4. The method for constructing an in situ biological 3D printed artificial liver patch for treating liver failure according to claim 1 or 3, characterized in that: It is characterized by: In step S2, the mixing volume ratio of the composite hydrogel and the mouse primary liver cells is 1:

1.

5. The method for constructing an in situ biological 3D printed artificial liver patch for treating liver failure according to claim 1, characterized in that: It is characterized by: In step S3, select the appropriate extrusion nozzle diameter, adjust the printing nozzle temperature to 10-15℃, adjust the printing platform temperature to 5-10℃, and use 2-4mm 3 The cell-containing bio-ink was printed in a 6 cm sterile culture dish at an extrusion speed of / s.

6. The method for constructing an in situ biological 3D printed artificial liver patch for treating liver failure according to claim 1, characterized in that: In step S4, the mass concentration of the CaCl2 solution is 3%.

7. The method for constructing an in situ biological 3D printed artificial liver patch for treating liver failure according to claim 1 or 6, characterized in that: In step S4, the cells were cultured in a 37° C., 5% CO 2 incubator using DMEM / F-12 medium containing acetylcysteine, nicotinamide, recombinant human growth factor, Wnt3a protein, 5% fetal bovine serum, and double-antibody, and the medium was changed regularly to maintain activity.

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