Construction method of 3D biological printing fatty liver tissue
By constructing fatty liver tissue using 3D bioprinting technology, the limitations of existing models in simulating the microenvironment and ethical constraints have been addressed. This has enabled a highly realistic research platform for fatty liver disease, improving the efficiency and success rate of drug development.
Patent Information
- Application Number
- CN202511321409.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-16
- Publication Date
- 2025-12-19
AI Technical Summary
Existing fatty liver models lack microenvironment simulation, animal models are subject to ethical and individual variability limitations, and organoids are small in size, expensive, and have low fidelity, making it difficult to meet the needs of highly realistic disease research.
Adipose liver tissue was constructed using 3D bioprinting technology. By preparing bio-ink and cross-linking it during printing, combined with in vitro culture and adjustment of culture medium composition, non-alcoholic and alcoholic fatty liver models were induced, achieving efficient and low-cost construction of large-size models.
It provides a highly realistic research platform for fatty liver disease, which is highly efficient, consistent, and accurate, enabling precise assessment of drug safety and efficacy, and shortening drug development time and costs.
Smart Images
Figure CN121160607A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of fatty liver model technology, and more specifically to a method for constructing 3D bioprinted fatty liver tissue. Background Technology
[0002] Currently, fatty liver disease is the leading cause of liver disease in my country, and in-depth research on it urgently requires highly realistic disease models. While existing cell models are convenient to operate, they lack microenvironment simulation; animal models are limited by ethical and individual variability constraints; and while organoids integrate three-dimensional functions, they are limited by their small size, high cost, and low degree of realism.
[0003] Bioprinting technology precisely constructs millimeter-scale multicellular spatial structures to actively simulate the pathological microenvironment of lipid accumulation in fatty liver, thereby overcoming the shortcomings of traditional models. This technology provides a highly biomimetic and standardized research platform for elucidating disease mechanisms and accelerating drug screening.
[0004] Therefore, developing a method for constructing 3D bioprinted fatty liver tissue to prepare a novel model that combines large size, low cost, high efficiency, and high reproducibility is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention
[0005] In view of this, the present invention provides a method for constructing 3D bioprinted adipose liver tissue.
[0006] To achieve the above objectives, the present invention adopts the following technical solution:
[0007] A method for constructing 3D bioprinted adipose liver tissue includes the following steps:
[0008] (1) 3D bioprinting of liver tissue:
[0009] At 37℃, a composite liver-like hydrogel was mixed with mouse hepatocytes to prepare the bio-ink required for printing; liver tissue was printed; the printed liver was placed in a 3% CaCl2 solution to rapidly cross-link, thus obtaining 3D bioprinted liver tissue.
[0010] (2) Inducing fatty liver tissue:
[0011] 3D bioprinted liver tissue was cultured in vitro, and after being cultured and induced to maturity using hepatocyte expansion medium, different types of fatty liver were induced by adjusting the composition of the medium, including non-alcoholic fatty liver and alcoholic fatty liver.
[0012] Furthermore, the non-alcoholic fatty liver induction described in step (2) is as follows:
[0013] Dissolve the oleic acid-palmitic acid mixture (1:1 by mass) in anhydrous ethanol to a concentration of 100 mg / mL, prepare an anhydrous ethanol storage solution, and store at -20°C.
[0014] Prepare a 10% (w / v) BSA-PBS solution, filter it using a 22µm filter, and store it at 4°C.
[0015] Thaw the anhydrous ethanol storage solution, preheat the BSA-PBS solution at 37°C, and dilute the anhydrous ethanol storage solution with BSA-PBS solution to prepare second storage solutions with different dilution ratios.
[0016] The second storage solution was added to the hepatocyte expansion culture medium at a volume ratio of 10%.
[0017] Depending on the dilution ratio of the second storage solution, the final concentration of the oleic acid-palmitic acid mixture in the hepatocyte expansion culture medium with a mass ratio of 1:1 is 100-1000 uM, and the induction time is 3-4 days.
[0018] Furthermore, the final concentration of the oleic acid-palmitic acid mixture is 200-800 μM.
[0019] Furthermore, the alcoholic fatty liver induction described in step (2) is as follows:
[0020] Prepare a liver parenchymal cell expansion medium containing ethanol, and prepare it fresh each time. The volume ratio of ethanol in the medium should be 1%-8%, and the induction time should be 6-9 days.
[0021] Furthermore, the volume ratio of ethanol in the culture medium is 2%-4%.
[0022] Furthermore, in step (1), primary hepatocytes are obtained by perfusion of mouse liver, and after expansion and culture, they are mixed with the composite liver-like hydrogel to obtain bio-ink;
[0023] The final concentration of mouse hepatocytes in the bioink was 4 × 10⁻⁶. 6 per mL.
[0024] Further, the preparation method of the composite liver-like hydrogel in step (1) is as follows: preheat the phosphate-buffered saline (PBS) solution to 50°C, weigh gelatin, sodium alginate and methacrylamide hyaluronic acid in a mass ratio of 10:2:1, prepare a mixture and add PBS solution, with final mass concentrations of 5%, 1%, and 0.5%, respectively; stir thoroughly at 50°C to prepare the composite liver-like hydrogel.
[0025] Furthermore, the method for 3D printing liver tissue in step (1) is as follows:
[0026] Place the prepared hydrogel containing liver parenchymal cells in a refrigerator at 4°C for 15-20 minutes; select a 21G extrusion nozzle, adjust the nozzle temperature to 15-18°C, and the platform temperature to 4-8°C; use a 5-6mm... 3 The bio-ink was printed into a sterile 6-well plate at an extrusion speed of / s, and the print was cured with 405nm ultraviolet light during the printing process.
[0027] Furthermore, the in vitro culture conditions for step (2) are: induction culture at 37℃ and 5% CO2, with the medium changed every 24 hours.
[0028] Furthermore, the components of the liver parenchymal cell expansion culture medium are:
[0029] N2 additive (1X), B27 (1X), 1mM acetylcysteine, 10mM nicotinamide, recombinant human growth factor: 2ng / ml FGF10, 50ng / mL EGF, 25ng / mL HGF, 10μMY-27632, 50ng / mL Wnt3a protein, 1% fetal bovine serum, 1% penicillin-streptomycin, and the remainder was Advanced DMEM / F-12 medium.
[0030] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows:
[0031] The artificial liver tissue described in this invention can be uniformly and standardizedly produced in vitro using 3D bioprinting technology, enabling high-throughput drug screening. In vitro, it exhibits specific gene and protein expression levels and functional phenotypes characteristic of mature hepatocytes. Successfully induced fatty liver models demonstrate disease characteristics corresponding to physiological pathology and respond sensitively to drugs. This invention overcomes the limitations of existing cell models (while convenient to operate, they lack microenvironment simulation), animal models (constrained by ethical and individual variability limitations), and organoids (while integrating three-dimensional functions, they are limited by small size, high cost, and low fidelity). The disease model constructed using 3D bioprinting technology provides a research platform for fatty liver disease that closely approximates the actual pathological state. Its advantages, including high efficiency, high consistency, high fidelity, and high sensitivity, facilitate deeper research into the disease mechanism, accurate and rapid assessment of drug safety and efficacy, and reduced drug development time and cost, bringing new hope for improving the success rate of new drugs in preclinical research. Attached Figure Description
[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.
[0033] Figure 1 Microscopic images of artificial liver tissue cultured in vitro for 0, 1, 4, and 7 days;
[0034] Figure 2 The results of Nile Red staining at different induction concentrations in a non-alcoholic fatty liver model;
[0035] Figure 3 BODITY staining results for an alcoholic fatty liver model at different induction concentrations;
[0036] Figure 4 The expression of genes related to fatty liver in a fatty liver model;
[0037] Figure 5 The therapeutic effect of statins on a model of non-alcoholic fatty liver disease. Detailed Implementation
[0038] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0039] Source of experimental materials:
[0040] N2 Additive (100X): Manufacturer: Gibco, Production Batch No. 17502048;
[0041] B27(100X): Manufacturer: Gibco, Batch No.: 17504044;
[0042] Y-27632: Manufacturer: MCE, Production Batch Number: HY-10071;
[0043] Advanced DMEM / F-12 culture medium: Manufacturer: Thermo, Batch number: c11330500BT.
[0044] Example 1
[0045] Primary hepatocytes were first obtained by perfusion of mouse livers. These cells were then expanded and cultured in hepatocyte expansion medium to reach a certain quantity. The in vitro culture conditions were: 37℃, 5% CO2, with medium changes every 24 hours. Mouse hepatocytes were imaged under a microscope after 1, 2, 3, 4, 5, 6, and 7 days of in vitro culture.
[0046] The components of the liver parenchymal cell expansion culture medium are:
[0047] N2 additive (1X), B27 (1X), 1mM acetylcysteine, 10mM nicotinamide, recombinant human growth factor: 2ng / ml FGF10, 50ng / mL EGF, 25ng / mL HGF, 10μMY-27632, 50ng / mL Wnt3a protein, 1% fetal bovine serum, 1% penicillin-streptomycin, and the remainder was Advanced DMEM / F-12 medium.
[0048] To prepare a composite liver-like hydrogel, phosphate-buffered saline (PBS) solution was preheated to 50°C. Gelatin, sodium alginate, and methacrylamide hyaluronic acid were weighed in a mass ratio of 10:2:1 and mixed. PBS solution was then added to the mixture, resulting in final mass concentrations of 5% gelatin, 1% sodium alginate, and 0.5% hyaluronic acid. The mixture was thoroughly stirred at 50°C to obtain the composite liver-like hydrogel.
[0049] At 37℃, a composite liver-like hydrogel was mixed with mouse hepatocytes to prepare the bio-ink required for printing; the final concentration of mouse hepatocytes in the bio-ink was 4×10⁻⁶. 6 per mL.
[0050] Bio-inks for printing were prepared by mixing a composite liver-like hydrogel with mouse hepatocytes.
[0051] Place the prepared hydrogel containing liver parenchymal cells in a refrigerator at 4°C for 18 minutes; select a 21G extrusion nozzle, adjust the nozzle temperature to 16°C, and the platform temperature to 5°C; use a 5mm... 3 The bio-ink was printed into a sterile 6-well plate at an extrusion speed of / s, and the print was cured with 405nm ultraviolet light during the printing process.
[0052] The printed liver was placed in a 3% CaCl2 solution to rapidly cross-link, resulting in 3D bioprinted liver tissue.
[0053] Figure 1 The results showed that hepatocytes maintained good cell viability, biological activity, and stability in the printed body, and could be used for subsequent disease induction and treatment.
[0054] 3D bioprinted liver tissue was cultured in vitro, and after being cultured and induced to maturity using hepatocyte expansion medium (about 1 week), different types of fatty liver were induced by adjusting the composition of the culture medium.
[0055] Non-alcoholic fatty liver induction: Dissolve a mixture of oleic acid and palmitic acid in anhydrous ethanol at a mass ratio of 1:1 to a concentration of 100 mg / mL, prepare an anhydrous ethanol storage solution, and store at -20℃.
[0056] Prepare a 10% (w / v) BSA-PBS solution, filter it using a 22µm filter, and store it at 4°C.
[0057] Thaw the anhydrous ethanol storage solution, preheat the BSA-PBS solution at 37°C, add the BSA-PBS solution to the anhydrous ethanol storage solution, dilute at different ratios, and prepare the second storage solution.
[0058] The second storage solution was added to the hepatocyte expansion culture medium at a volume ratio of 10%.
[0059] The final concentration of the oleic acid-palmitic acid mixture is determined based on the dilution ratio of the second storage solution. (For example, to prepare 20 ml of induction medium with a final concentration of 100 μM for the oleic acid-palmitic acid mixture, 5.39 μL of anhydrous ethanol storage solution needs to be dissolved in 200 μL of 10% (w / v) BSA-PBS solution to prepare the second storage solution, which is then dissolved in 1.8 ml of hepatocyte expansion medium.)
[0060] The final concentrations of the oleic acid-palmitic acid mixture (1:1 mass ratio) in the hepatocyte expansion medium were set into three groups:
[0061] 0 uM (normal control group), 200 uM, 400 uM, 800 uM;
[0062] Four days after in vitro induction of the artificial non-alcoholic fatty liver model, Nile Red staining was performed on it.
[0063] Figure 2 The results showed that the accumulation of lipid droplets in the cells of the induced artificial liver tissue increased significantly, and the accumulation of lipid droplets increased with the increase of the induction concentration, thus aggravating the degree of fatty liver. The artificial non-alcoholic fatty liver model was successfully constructed.
[0064] Alcoholic fatty liver induction: Prepare liver parenchymal cell expansion medium containing ethanol, freshly prepared and used immediately. The volume ratio of ethanol in the medium was set to three groups:
[0065] 0% (normal control group), 2%, 4%;
[0066] Seven days after in vitro induction of the artificial alcoholic fatty liver model, Nile Red staining was performed on it.
[0067] Figure 3 The results showed that the accumulation of lipid droplets in the cells of the induced artificial liver tissue increased significantly, and the accumulation of lipid droplets increased with the increase of the induction concentration, thus aggravating the degree of fatty liver. The artificial alcoholic fatty liver model was successfully constructed.
[0068] The mRNA expression levels of fatty liver-related genes in artificial liver tissues before and after induction were detected in two groups:
[0069] 3D bioprinted liver tissue (control group), artificial non-alcoholic fatty liver model (400uM);
[0070] Figure 4 The results showed that the expression of fatty liver-related genes (Cd36, Fatp2, Fasn, Scd1) was upregulated in the induced artificial liver tissue, confirming the successful construction of the fatty liver disease model at the gene expression level.
[0071] An artificial non-alcoholic fatty liver disease model (400uM group) was treated with statins and divided into three groups:
[0072] 0 mM (normal control group), 0.1 mM, 10 mM;
[0073] The therapeutic effect was observed 48 hours later using oil red chromosome analysis.
[0074] Figure 5 The results showed that statin treatment reduced intracellular lipid droplet accumulation in the artificial non-alcoholic fatty liver model, and the reduction became more pronounced with increasing drug concentration, confirming the model's sensitive response to statins and demonstrating the significant therapeutic effect of statins on non-alcoholic fatty liver.
[0075] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those 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 invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. A method for constructing 3D bioprinted adipose liver tissue, characterized in that, Includes the following steps: (1) 3D bioprinting of liver tissue: At 37℃, a composite liver-like hydrogel was mixed with mouse hepatocytes to prepare the bio-ink required for printing; liver tissue was printed; the printed liver was placed in a 3% CaCl2 solution to rapidly cross-link, thus obtaining 3D bioprinted liver tissue. (2) Inducing fatty liver tissue: 3D bioprinted liver tissue was cultured in vitro, and after being cultured and induced to maturity using hepatocyte expansion medium, different types of fatty liver were induced by adjusting the composition of the medium, including non-alcoholic fatty liver and alcoholic fatty liver.
2. The construction method according to claim 1, characterized in that, Step (2) describes the induction of non-alcoholic fatty liver disease: Dissolve the oleic acid-palmitic acid mixture (1:1 by mass) in anhydrous ethanol to a concentration of 100 mg / mL, prepare an anhydrous ethanol storage solution, and store at -20°C. Prepare a 10% (w / v) BSA-PBS solution, filter it using a 22µm filter, and store it at 4°C. Thaw the anhydrous ethanol storage solution, preheat the BSA-PBS solution at 37°C, and dilute the anhydrous ethanol storage solution with BSA-PBS solution to prepare second storage solutions with different dilution ratios. The second storage solution was added to the hepatocyte expansion culture medium at a volume ratio of 10%. Depending on the dilution ratio of the second storage solution, the final concentration of the oleic acid-palmitic acid mixture in the hepatocyte expansion culture medium with a mass ratio of 1:1 is 100-1000 uM, and the induction time is 3-4 days.
3. The construction method according to claim 2, characterized in that, The final concentration of the oleic acid-palmitic acid mixture is 200-800 μM.
4. The construction method according to claim 1, characterized in that, The alcoholic fatty liver induction described in step (2): Prepare a liver parenchymal cell expansion medium containing ethanol, and prepare it fresh each time. The volume ratio of ethanol in the medium should be 1%-8%, and the induction time should be 6-9 days.
5. The construction method according to claim 4, characterized in that, The ethanol volume ratio in the culture medium is 2%-4%.
6. The construction method according to claim 1, characterized in that, In step (1), primary hepatocytes are obtained by perfusion of mouse liver, and after expansion and culture, they are mixed with the composite liver-like hydrogel to obtain bio-ink; The final concentration of mouse hepatocytes in the bioink was 4 × 10⁻⁶. 6 per mL.
7. The construction method according to claim 1, characterized in that, The method for preparing the composite liver-like hydrogel in step (1) is as follows: preheat the phosphate-buffered saline (PBS) solution to 50°C, weigh gelatin, sodium alginate and methacrylamide hyaluronic acid in a mass ratio of 10:2:1, prepare a mixture and add PBS solution, with final mass concentrations of 5%, 1% and 0.5% respectively; stir thoroughly at 50°C to prepare the composite liver-like hydrogel.
8. The construction method according to claim 1, characterized in that, The method for 3D printing liver tissue in step (1) is as follows: Place the prepared hydrogel containing liver parenchymal cells in a refrigerator at 4°C for 15-20 minutes. Select a 21G extrusion nozzle, adjust the nozzle temperature to 15-18℃, and the platform temperature to 4-8℃; use a 5-6mm nozzle. 3 The bio-ink was printed into a sterile 6-well plate at an extrusion speed of / s, and the print was cured with 405nm ultraviolet light during the printing process.
9. The construction method according to claim 1, characterized in that, Step (2) In vitro culture conditions are: induction culture at 37℃ and 5% CO2, with medium changed every 24 hours.
10. The construction method according to claim 1, characterized in that, The components of the liver parenchymal cell expansion culture medium are: N2 additive (1X), B27 (1X), 1mM acetylcysteine, 10mM nicotinamide, recombinant human growth factor: 2ng / ml FGF10, 50ng / mL EGF, 25ng / mL HGF, 10μMY-27632, 50ng / mL Wnt3a protein, 1% fetal bovine serum, 1% penicillin-streptomycin, and the remainder was Advanced DMEM / F-12 medium.