Construction method and application of 3D printing liver organoid based on hiHeps

Abstract

The invention belongs to the technical field of biological manufacturing and regenerative medicine, and discloses a cell construction method based on three-dimensional biological printing. The method comprises the following steps: dissolving GelMA and a photoinitiator LAP in a hepatocyte maintenance medium to prepare bio-ink; after filtration and sterilization, human induced hepatocytes are digested, counted and resuspended in the bio-ink according to the set cell density; and loading the obtained mixed solution to a 3D printing nozzle with temperature control and extrusion control, carrying out layer-by-layer printing at a set temperature and an extrusion rate, and carrying out photo-crosslinking curing to form the cell construct. According to the method, high-precision control and high cell survival rate in the printing process can be achieved, and the method is suitable for rapid prototyping preparation of various cell-containing biological materials. The technology has the advantages of being easy and convenient to operate, high in material universality, high in expandability and the like, and a universal manufacturing scheme can be provided for construction of tissue engineering and drug screening models.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of 3D printing liver organoids, and particularly relates to a construction method and application of a 3D printing liver organoid based on hiHeps. BACKGROUND

[0002] The liver is an important organ in the human body that undertakes metabolism, synthesis and detoxification, and function damage of the liver can cause serious diseases such as acute or chronic liver failure. At present, clinical treatment mainly relies on liver transplantation, but is limited by problems such as donor shortage and immune rejection. A bioartificial liver system (BAL) can temporarily replace part of the liver function, but the core lies in the source of functional hepatocytes with metabolic activity. In recent years, human induced hepatocytes (hiHeps) have become an ideal cell source due to their amplification, low immunogenicity and strong metabolic activity. However, the traditional two-dimensional culture system is difficult to maintain the specific function of hiHeps, which limits the application of hiHeps in in vitro liver function reconstruction and BAL systems. Three-dimensional bioprinting technology (3D printing) can realize the biomimetic construction of liver tissue by precisely controlling the spatial distribution of cells and the composition of the microenvironment. Studies have shown that a matrix of methacrylate gelatin (GelMA) and a light initiator lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate (LAP) has good biocompatibility, but the system optimization of the printing conditions and material system for hiHeps has not been disclosed. The application proposes a new three-dimensional bioprinting method for efficiently constructing a functional and stable liver tissue model. SUMMARY

[0003] In view of the above problems, the application aims to provide a construction method of a 3D printing liver organoid based on hiHeps, which constructs a biomimetic liver tissue or organoid with biological activity by three-dimensional bioprinting technology. To achieve the above-mentioned purpose, the technical scheme adopted by the application is as follows: The application provides a method for 3D printing a liver organoid, and the liver organoid cell biological ink is obtained by 3D printing, wherein the cell biological ink comprises hiHep cells and a prefabricated biological ink.

[0004] In this invention, the term "cell bio-ink" refers to a cell-biomaterial hybrid system used in biomanufacturing that maintains fluidity under printing conditions and forms a three-dimensional support structure after printing, exhibiting good biocompatibility, printability, and cell viability retention. The term "pre-prepared bio-ink" refers to a cell-free bio-ink substrate material that is pre-prepared, formulated, and prepared to a printable state before the addition of cells, typically comprising a printable matrix material, a cross-linking system, a culture medium, or a buffer solution.

[0005] Furthermore, the pre-made bio-ink includes natural polymer materials, synthetic polymer materials, composite hydrogel materials, and extracellular matrix-derived materials; the natural polymer materials include gelatin, methacrylamide gelatin (GelMA), sodium alginate, collagen, hyaluronic acid, and silk fibroin; the synthetic polymer materials include polyethylene glycol diacrylate (PEGDA) and polyvinyl alcohol (PVA); the composite hydrogel materials include GelMA / Alginate composite gel, GelMA / PEGDA composite gel, Alginate / Matrigel mixed hydrogel, etc.; the extracellular matrix-derived materials include decellularized matrix hydrogel (dECM), Matrigel, etc.

[0006] Furthermore, the pre-made bio-ink is a natural polymer material.

[0007] Furthermore, the natural polymer material is GelMA.

[0008] Furthermore, the concentration of the GelMA is 3% to 10%.

[0009] Furthermore, the concentration of the GelMA is 5%.

[0010] Further, the pre-prepared bio-ink further comprises a cross-linking initiator; the cross-linking initiator comprises a photo-cross-linking initiator, an ionic cross-linking initiator, an enzymatic cross-linking initiator, a temperature-sensitive cross-linking initiator; the photo-cross-linking initiator comprises Lithium Phenyl(2,4,6-Trimethylbenzoyl)phosphinate (LAP), 2-Hydroxy-1-[4-(hydroxyethoxy)phenyl]-2-methyl-1-propanone (Irgacure 2959); the ionic cross-linking initiator comprises calcium chloride (CaCl2), sodium chloride (NaCl), etc.; the enzymatic cross-linking initiator comprises hydrogen peroxide (H2O2), a Horseradish Peroxidase (HRP) system; the temperature-sensitive cross-linking initiator comprises a thermoresponsive polymer such as poly(N-isopropylacrylamide) (PNIPAAm) or gelatin.

[0011] Further, the cross-linking initiator is a photo-cross-linking initiator.

[0012] Further, the photo-cross-linking initiator is LAP.

[0013] Further, the concentration of the LAP is 0.05% to 0.5%.

[0014] Further, the concentration of the LAP is 0.25%.

[0015] Further, the pre-prepared bio-ink further comprises a hepatocyte maintenance medium.

[0016] In a specific embodiment of the present application, the cell bio-ink comprises 5% GelMA, 0.25% LAP, and hiHep cells.

[0017] Further, the 3D printing specifically comprises the following operation: placing the bio-ink of the first aspect of the present application in a nozzle of a 3D printing device, and performing layer-by-layer extrusion printing.

[0018] Further, the nozzle comprises an extrusion nozzle, a jetting nozzle, and a light-curing nozzle.

[0019] Further, the nozzle is an extrusion nozzle.

[0020] Further, the extrusion nozzle comprises a pneumatic drive nozzle, a screw drive nozzle, and a piston drive nozzle; the jetting nozzle comprises a piezoelectric drive nozzle, a thermal bubble nozzle, and an electromagnetic drive nozzle; the light-curing nozzle comprises a digital light processing nozzle, a stereolithography nozzle, and a laser scanning light-curing nozzle.

[0021] Further, the extrusion type nozzle is a gas pressure driven nozzle.

[0022] Further, the gas pressure driven nozzle comprises a syringe barrel, a needle, a driving module, and a temperature control module.

[0023] In the present application, the term "driving module" refers to a control component that controls the extrusion rate and volume flow of biological ink through gas pressure, mechanical or electrical driving force, including pressure regulating valve, air pump, solenoid valve and electronic control unit connected therewith; the term "temperature control module" refers to a device for adjusting the temperature of the nozzle and biological ink to maintain the printing rheological properties and cell activity, including refrigeration cycle system, thermoelectric cooling sheet, temperature sensor and control unit.

[0024] Further, the needle is 27G-33G.

[0025] In a specific embodiment of the present application, the needle is 32G.

[0026] Further, the temperature control module sets the temperature to 10-20℃.

[0027] In a specific embodiment of the present application, the temperature control module sets the temperature to 11℃.

[0028] Further, the method further comprises photo-crosslinking curing and culture, specifically comprising the following operations: irradiating the printing result by a light source to realize photo-crosslinking curing, the irradiation time being 30-60 s, transferring the crosslinked structure to a hepatocyte maintenance culture medium, and replacing the culture medium every two days.

[0029] Further, the light source is ultraviolet light, visible blue light, and near ultraviolet long wave.

[0030] Further, the commonly used irradiation wavelength of the ultraviolet light is 320-405 nm; the irradiation wavelength of the visible blue light is 405-480 nm; and the irradiation wavelength of the near ultraviolet long wave is 365-405 nm.

[0031] Further, the light source is visible blue light.

[0032] In a specific embodiment of the present application, the irradiation wavelength of the visible blue light is 405 nm.

[0033] The second aspect of the present application provides a raw material for printing liver organoids, the raw material comprising the hiHep cells and / or the pre-made bioink of the first aspect of the present application. In the present application, the term "and / or" means that either of the objects can be independently included, or multiple objects can be simultaneously included. Specifically, "hiHep cells and / or pre-made bioink" includes the following combinations: only hiHep cells, only pre-made bioink, or both hiHep cells and pre-made bioink.

[0034] Further, the hiHep cells are harvested from the hepatocyte maintenance medium.

[0035] In the present application, the term "hepatocyte maintenance medium" refers to a culture system that can support the long-term culture of human induced hepatocytes (hiHeps) and maintain their specific liver functions, usually adding various growth factors, hormones and nutritional supplements to simulate the physiological microenvironment of hepatocytes in vivo.

[0036] Further, the hepatocyte maintenance medium components include basal medium, functional additive components.

[0037] Further, the basal medium includes DMEM / F12, 10% fetal bovine serum.

[0038] Further, the functional additive components include growth factors, hormones, trace nutrient supplements, neuronal supplements, and maintenance additives; the growth factors include epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), hepatocyte growth factor (HGF); the hormones include dexamethasone, insulin; the trace nutrient supplements include transferrin, sodium selenite, and their complexes (collectively referred to as insulin-transferrin-selenium, ITS); the neuronal supplements include SM1 Neuronal Supplement, B27 Supplement, or N2 Supplement; the maintenance additives include nicotinamide, ascorbic acid, or a mixture of non-essential amino acids.

[0039] Further, the hepatocyte maintenance medium components include any one of insulin-transferrin-selenium, SM1 neuronal supplement, TGF-alpha, EGF, or dexamethasone.

[0040] In specific embodiments of the present application, the components of the hepatocyte maintenance medium are insulin-transferrin-selenium, SM1 neural cell supplement, 40 ng / mL TGF-alpha, 40 ng / mL EGF, and 10 μM dexamethasone.

[0041] Further, the hiHep cells are resuspended into a cell suspension after trypsin digestion.

[0042] Further, the cell concentration of the cell suspension is 2×10 7 ~ 6×10 7 cells / mL.

[0043] Further, the pre-prepared bio-ink comprises GelMA and LAP.

[0044] Further, the concentration of the GelMA is 3%~10%.

[0045] Further, the concentration of the GelMA solution is 5%.

[0046] Further, the concentration of the LAP is 0.05%~0.5%.

[0047] Further, the concentration of the LAP is 0.25%.

[0048] Further, the pre-prepared bio-ink further comprises a hepatocyte maintenance medium.

[0049] In specific embodiments of the present application, the hiHep cells are suspended in the bio-ink at a cell density of 1×10 7 or 5×10 7 cells / mL.

[0050] In a third aspect, the present application provides a liver organoid or tissue, which is prepared from the method of the first aspect or the raw material of the second aspect.

[0051] In the present application, the term "liver organoid or tissue" refers to a system formed in vitro by three-dimensional bioprinting technology, using human induced hepatocytes (hiHeps) as the main cell source, in a biocompatible scaffold material (such as GelMA, Matrigel, or acellular matrix hydrogel). The system has three-dimensional structural characteristics and partial liver-specific functions. The system can maintain cell activity and metabolic function for a long period in vitro, and can partially simulate the physiological function and pathological response of the liver, and is used for disease model construction, drug screening, or biological artificial liver function module research.

[0052] In a fourth aspect, the present application provides any one of the following uses: (1) the method of the first aspect, the raw material of the second aspect, the liver organoid or model of the third aspect in the application of 3D printing liver organoid or tissue; (2) the method of the first aspect, the raw material of the second aspect, the liver organoid or model of the third aspect in the application of evaluating the efficacy of drugs for treating liver-related diseases; (3) the method of the first aspect, the raw material of the second aspect, the liver organoid or tissue of the third aspect in the application of constructing an in vitro liver function reconstruction system; (4) the method of the first aspect, the raw material of the second aspect, the liver organoid or tissue of the third aspect in the application of constructing a bioartificial liver system.

[0053] Further, the diseases include any one or more of acute liver injury, drug-induced liver injury, acute or chronic liver failure, liver fibrosis, liver cirrhosis, non-alcoholic fatty liver disease, genetic metabolic liver disease, cholestatic hepatitis, or hepatocellular carcinoma.

[0054] In the present application, the term "in vitro liver function reconstruction system" refers to an experimental system that realizes the reconstruction of main physiological functions of the liver (such as metabolism, synthesis, detoxification) under in vitro conditions through the synergistic effect of cells, biomaterials and culture environment.

[0055] Further, the liver functions include protein synthesis function, ammonia metabolism function, drug metabolism function, bilirubin metabolism function, energy metabolism function, and toxicity response function.

[0056] Further, the bioartificial liver system includes a bioreactor module, an extracorporeal circulation module, and an auxiliary module. The term "artificial biological liver system" refers to an in vitro auxiliary device constructed based on the principle of cell bioreactor, which performs biochemical metabolism and detoxification treatment on patient's plasma or circulating fluid through a bioreactor containing active liver cells or organoids, thereby partially replacing the damaged liver function.

[0057] Further, the bioreactor module includes a hollow fiber membrane reactor, a fixed bed or a microfluidic chip structure; the extracorporeal circulation module includes a plasma separation device, a peristaltic pump, a connecting pipeline, a flow rate and pressure control unit; the auxiliary module includes a temperature control system, an oxygen supply adjusting device, a sensing detection module (detecting pH, oxygen partial pressure, ammonia concentration, urea concentration, etc.), a safety alarm system.

[0058] The present application has the advantages and beneficial effects: (1) Cell source is safe and function is stable. The present application adopts human induced hepatocytes (Human Induced Hepatocytes, hiHeps) as the printing cell source, which is stable, can be amplified, has no risk of tumor formation, can maintain long-term albumin secretion, urea synthesis and cytochrome P450 enzyme activity in vitro, and has good functional maturity.

[0059] (2) The printing system has high biocompatibility and good forming precision. The present application adopts a biological ink composed of methacrylated gelatin (GelMA) and lithium phenyl (2,4,6-trimethylbenzoyl) phosphonate (LAP), which has good rheological properties and photocrosslinking stability, and can form a three-dimensional construct with a bionic liver lobule structure after printing.

[0060] (3) The printing parameters are optimized, and the cell survival rate is high. By using a gas pressure driven extrusion nozzle and a 32G needle, and printing under temperature control conditions of 10-20℃, the risk of cell damage by shear stress is effectively reduced, ensuring high survival and uniform distribution of cells after printing.

[0061] (4) The function of the construct can be used for multi-field verification. The three-dimensional liver organoid prepared can not only maintain liver function indicators in vitro, but also can be further used for drug metabolism, toxicology evaluation and bioartificial liver (Bioartificial Liver, BAL) system module construction.

[0062] (5) The method is versatile and expandable. The printing system and process conditions of the present application are suitable for various types of cells and composite hydrogel materials, and can be extended to other tissue engineering printing fields such as kidney and pancreas, and have good technical universality and industrialization prospects. BRIEF DESCRIPTION OF DRAWINGS

[0063] Figure 1 The construction process and application scenarios of three-dimensional bioprinted human hepatocyte organs; Figure 2Figure 1. Partitioned features of three-dimensional bioprinted human liver organoids; wherein, panel A is representative live / dead cell assay of hiHep cells in two-dimensional and three-dimensional culture, scale bar: 200 pm; panel B is quantitative analysis of survival rate of hiHep cells in two-dimensional and three-dimensional culture (n=3 per group); panel C is survival rate of 2D cultured hiHep cells and 3D P-HHO cells detected by CCK-8 method (n=3 per group); panel D is morphological comparison of 2D cultured hiHep cells, 3D bioprinted hiHeps organoids and 3D P-HHO models after 1 week / month of culture (red arrows: liver cord-like structures formed by self-assembly; white arrows: hepatocyte spheroids spontaneously formed by hiHeps), scale bar: 50 pm; panel E is distribution of differentially expressed genes in 3D P-HHO and 2D cultured hiHep cells, key liver genes are highlighted; panel F is KEGG enrichment analysis of upregulated genes, revealing their biological processes related to liver functions; panel G is heatmap of partition marker gene expression in HepRG, hiHeps, 3D P-HHO and primary human hepatocytes (PHH) (showing gene expression difference between central and periportal hepatocytes); panel H is schematic diagram of spatial distribution of centrilobular (red) and perisinusoidal (green) marker gene expression in healthy liver; panel I is representative immunofluorescence images showing distribution of CYP3A4 (red), HAL (green) and DAPI (blue) in healthy mouse liver and 3D P-HHO models, the structural and functional features of which are highly similar to native liver tissue (white arrow: CYP3A4 positive area, indicating periportal hepatocytes; white pentagon: HAL positive area, indicating periportal hepatocytes), scale bar: 100 pm for mouse liver; 25 pm for 3D P-HHO models; Figure 3Figure 6. Functional characterization of 3DP-HHOs; wherein Figure A is a heatmap distribution of glucose metabolism related genes in HepRG, hiHeps, 3DP-HHOs and PHHs; Figure B is a heatmap distribution of lipid metabolism related genes in HepRG, hiHeps, 3DP-HHOs and PHHs; Figure C is a heatmap distribution of amino acid metabolism related genes in HepRG, hiHeps, 3DP-HHOs and PHHs; Figure D is PAS staining of 2D cultured hiHeps and 3DP-HHOs, scale bars are 200 pm and 500 pm, respectively; Figure E is Oil Red staining results of 2D cultured hiHep cells treated with BSA or OA and 3DP-HHOs treated cells, scale bar: 50 pm; Figure F is the comparison of ALB and urea nitrogen concentrations in 2D cultured hiHeps and 3DP-HHOs cell culture supernatants (n=3 for each group); Figure G is a heatmap distribution of drug metabolism related genes in HepRG, hiHeps, 3DP-HHOs and PHHs; Figure H is a heatmap distribution of bile secretion and bilirubin metabolism related genes in HepRG, hiHeps, 3DP-HHOs and PHHs; Figure I is Dil-Ac-LDL uptake experiment of 2D cultured hiHeps and 3DP-HHOs cells, scale bars: 20 pm, 100 pm and 2 mm; Figure J is ICG uptake and release experiment of 2D cultured hiHeps and 3DP-HHOs cells, scale bar: 500 pm; Figure 4Fig. 3D is a representative histological image of each experimental group of TAA-induced liver fibrosis, stained with HE, Masson and Sirius red, scale bar: 200 pm and 250 pm; Fig. 3E is a schematic diagram of a carbon tetrachloride (CCl4)-induced liver fibrosis model and experimental grouping: sham operation group (Sham), 3DP-GelMA group, hiHeps group and 3DP-HHO group (n=5 in each group); Fig. 3F is a quantitative analysis of the serum albumin level of each experimental group in the CCl4-induced liver fibrosis model (n=5 in each group); Fig. 3G is a representative histological image of each experimental group of CCl4-induced liver fibrosis, stained with HE, Masson and Sirius red, scale bar: 200 pm and 250 pm; Fig. 3H is a Western blotting method for detecting the expression level of a-SMA in the liver tissue lysate of each experimental group in the CCl4-induced liver fibrosis model, using tubulin as an internal control (n=3 in each group); Fig. 3I is a quantitative PCR analysis of fibrosis-related genes (TGF-β, a-SMA, Timp1, Col1a1) (n=5 in each group); Figure 5Results of 3DP-HHOs to promote liver regeneration and alleviate APAP-induced acute liver injury; wherein, panel A is a schematic diagram of the experimental procedure: after intraperitoneal injection of 300 mg / kg APAP to induce liver injury in Balb / c nude mice, 3D-printed liver organoids (3DP-HHOs) with different densities (0.5M-5M cells) were implanted, with 5 mice in each group; panel B is the detection of serum ALT levels at 24 hours and 48 hours in each experimental group in the APAP-induced acute liver failure model (n=5 in each group); panel C is the representative H&E staining results of liver sections at 24 hours and 48 hours after intraperitoneal injection of APAP in the APAP-induced acute liver failure model; scale bar: 500 µm; panel D is the Ki67 immunohistochemical staining results of liver sections at 24 hours and 72 hours after intraperitoneal injection of APAP in the APAP-induced acute liver failure model, scale bar: 100 µm; panel E is the Western blot analysis of regeneration-related proteins (PCNA, CDK4, CDK1, pH3S10) at 72 hours in each experimental group in the APAP-induced acute liver failure model (n=3 in each group); panel F is the quantitative analysis of necrotic area (%) by HE staining in each experimental group in the APAP-induced acute liver failure model (n=5 in each group); panel G is the quantitative analysis of Ki67-positive nuclei at 24 hours and 72 hours in each experimental group in the APAP-induced acute liver failure model (n=5 in each group); panel H is the densitometric analysis of the Western blot detection results in each experimental group in the APAP-induced acute liver failure model (n=3 in each group); Figure 6Figure 3. 3DP-HHOs can alleviate hepatocyte injury and promote hepatocyte regeneration in a CCl4-induced acute liver failure model. A, Timeline of the experiment: Balb / c nude mice were given 20 mL / kg of 10% CCl4intraperitoneally to induce liver injury, and then 3D-printed liver organoids (3DP-HHOs) were implanted at different densities (0.5M-5M cells) (n=5 for each group). B, Serum ALT levels were detected at 24 h and 48 h in the CCl4-induced acute liver failure model (n=5 for each group). C, Liver sections were observed by HE and TUNEL staining at 24 h and 48 h after APAP-induced acute liver failure in different groups. The scale bars are 500 µm and 50 µm, respectively. D, Representative Ki67 immunohistochemical staining of liver sections at 24 h and 72 h after intraperitoneal injection of CCl4in the CCl4-induced acute liver failure model. The scale bar is 50 µm. E, Western blot analysis of regeneration-related proteins (PCNA, CDK4, CDK1, and pH3S10) at 72 h in the CCl4-induced acute liver failure model (n=3 for each group). Figure 7 Figure 4. 3DP-HHOs can promote hepatocyte proliferation in a partial hepatectomy (PHx) model. A, Schematic diagram of the experimental procedure: Balb / c nude mice underwent 2 / 3 partial hepatectomy, and then 3D-printed liver organoids (3DP-HHOs) or solvent controls were implanted. Liver tissues and serum samples were collected at 24 h, 48 h, and 96 h after the operation (n=5 for each group). B, Liver-to-body weight ratios of the experimental groups in the PHx model (n=5 for each group). C, Serum ALT and AST levels of the experimental groups in the PHx model (n=5 for each group). D, Representative H&E staining and Ki67 immunohistochemical staining of liver sections at 24 h and 48 h after the operation in the PHx model. The scale bars are 50 µm and 200 µm, respectively. E, Quantitative analysis of Ki67-positive nuclei in liver sections at 24 h and 48 h after the operation in the PHx model (n=5 for each group). F, Western blot analysis of regeneration markers (PCNA, CDK4, CDK1, and pH3S10) at 24 h and 48 h after the operation in the PHx model (n=3 for each group). G, qRT-PCR detection of cell cycle protein gene (Cyclin D1, B1, A2, and E1) expression levels at 24 h and 48 h after the operation in the PHx model (n=5 for each group). H, Density analysis of Western blot detection in the PHx model (n=3 for each group). Figure 8Results of 3DP-HHO to promote liver regeneration through upregulation of matrix metalloproteinases (MMPs) and HGF signaling pathways, and to improve survival in patients with acute liver failure (PHLF); Panel A, qRT-PCR analysis of MMP2 and MMP9 in 2D cultured hiHeps and 3DP-HHO cells (n=3 per group); Panel B, ELISA analysis of culture supernatants of 2D cultured hiHep cells and 3DP-HHO cells (n=4 per group); Panel C, ELISA analysis of serum samples collected at 0, 24, and 48 hours post-surgery in the PHx model; Panel D, representative images of Transwell assay showing migration of 2D cultured hiHeps and 3D cultured HHO cells, scale bar: 250 µm; Panel E, representative immunofluorescence images showing human-specific GAPDH (red), ALB (green), and DAPI (blue) in liver sections at day 0 and day 7 post-implantation of 3DP-HHO, and in sham-operated group, scale bar: 100 µm; Panel F, representative immunofluorescence images showing cyclin D1 (red) and DAPI (blue) in liver sections at 24 and 48 hours post-surgery in the partial hepatectomy model; Panel G, Transwell assay to measure migration of 2D cultured hiHeps and 3DP-HHO cells (n=3 per group); Panel H, quantification of cyclin D1 -positive nuclei in liver sections at 24 and 48 hours post-surgery in the PHx model (n=5 per group); Panel I, survival analysis of patients with 85% partial hepatectomy with and without 3DP-HHO implantation; Panel J, survival analysis of patients with 90% partial hepatectomy with and without 3DP-HHO implantation; Panel K, mechanism of 3DP-HHO to achieve liver regeneration through promotion of hepatocyte migration and HGF / MMP-mediated extracellular matrix remodeling; Figure 9Figure A is the recovery of vitality in 85% partial hepatectomy patients before and after implantation of 3DP-HHO; Figure B is the Kaplan-Meier survival curve of 85% partial hepatectomy patients with or without implantation of 3DP-HHO (control group n=4, 3DP-HHO treatment group n=5); Figure C is the macroscopic observation of the liver residual tissue (caudate lobe) before and 24 hours after 85% partial hepatectomy, the time points include 24 hours and 168 hours after implantation of 3DP-HHO (yellow pentagram marks the implanted 3DP-HHO), the scale: 1 cm; Figure D is the computed tomography (CT) performed before and after 85% partial hepatectomy (PHx) and the 3D reconstruction of 3DP-HHO scanning performed 168 hours after surgery, the outline of the pig liver is marked with a red dashed line frame, and the implanted 3DP-HHO is marked with a yellow arrow; Figure E is the H&E staining of liver tissue implanted with 3DP-HHO, observed at 24, 36, 72 and 168 hours after 85% hepatectomy (dashed line separates liver tissue and 3DP-HHO; white asterisk marks liver tissue, yellow asterisk marks 3DP-HHO), scale: 50 µm; Figure F is the serum biochemical index of 85% partial hepatectomy patients with and without implantation of 3DP-HHO after surgery (control group n=4; 3DP-HHO treatment group n=2-5); Figure G is the results of HE staining and Ki67 immunohistochemical staining of liver tissue sections of 85% partial hepatectomy patients with and without implantation of 3DP-HHO at 24, 48, 72 and 120 hours after surgery, scale: 500 µm and 100 µm. DETAILED DESCRIPTION

[0064] The application will be further described below in conjunction with the examples. The following description is only the preferred embodiment of the application, and does not limit the application in other forms. Any skilled person in the art can modify the above disclosed technical content to equivalent embodiments. Any simple modification or equivalent change of the following examples without departing from the technical essence of the application falls within the protection scope of the application.

[0065] In the embodiments of the application, the primers and sequence information used in mouse RT-qPCR are shown in Table 1, the primary antibody information involved in immunofluorescence is shown in Table 2, and the antibody information related to Western blot experiment is shown in Table 3.

[0066] Table 1 RT-qPCR primer information table

[0067]

[0068] Table 2. Information of primary antibodies for immunofluorescence

[0069] Table 3. Information of antibodies for Western blot

[0070] In the embodiments of the present application, the statistical analysis method involved is: using GraphPad Prism software (version 10.1.2, USA) for statistical analysis. The data are expressed as mean ± standard deviation. The quantitative data are statistically analyzed by Student t test, one-way analysis of variance and Log-rank (Mantel-Cox) test. The significance levels are marked as follows: p0.05 (*), p<0.01 (**), p<0.001 (***), p<0.0001 (****).

[0071] Example 1: Preparation of hiHep three-dimensional bioprinting constructs (3DP-HHOs) The establishment, expansion and cryopreservation of hiHep cells were carried out according to the method in the literature “Reversal of liver failure using a bioartificial liver device implanted with clinical-grade human-induced hepatocytes”. Then the cells were cultured in hepatocyte maintenance medium (HMM) containing supplement components. The specific components of the culture medium are shown in Table 4. The hiHep cells used in downstream applications were within 10 generations to ensure cell integrity and functional activity.

[0072] Table 4. Specific components of HMM culture medium

[0073] Methacrylate gelatin (GelMA, 5% w / v, EFL-GM-60, EFL) and phenyl (2,4,6- trimethylbenzoyl) phosphate lithium salt (LAP, 0.25% w / v, EFL-LAP, EFL) were dissolved in hepatocyte maintenance medium (HMM) and stirred gently at 37 °C for 1 h. The obtained solution was filtered and sterilized by 0.22 μm Rapid-Flow disposable filter (BS-PES-22, Biosharp) to obtain a pre-prepared bio-ink.

[0074] hiHep cells digested with trypsin-EDTA (Gibco) were counted using a cell counter (Countess3 FL, Thermo Fisher Scientific) and then analyzed at a final concentration of 1×10⁻⁶. 7 and 5×10 7 Cells / mL were resuspended in pre-prepared bio-ink to obtain cell bio-ink.

[0075] Cell bio-ink was loaded into a 3D cell printer (Envision TEC 3D Bioplotter) and extruded layer by layer at a speed of 150 mm³ / min. After printing, the cells were photocrosslinked under 405 nm light. The resulting three-dimensional constructs (3DP-HHOs) were cultured in HMM medium with medium changes every two days. Cell proliferation activity was assessed using the Cell Counting Kit-8 (catalog number: 40203ES76, Yeasen). After culturing hiHep cells and 3DP-HHOs under the experimental conditions described above, they were incubated with CCK-8 solution diluted 1:10 in HMM medium at 37°C in the dark for 1 hour. The absorbance was then measured at 450 nm using a microplate reader. Cell viability was assessed according to the manufacturer's operating procedures of the Cell Viability and Toxicity Assay Kit (catalog number: C2015L, Beyotime, China). Calcein-AM was diluted with propidium iodide (PI) at a ratio of 1:1000, and after staining for 300 minutes, the samples were imaged using a fluorescence microscope (EVOS FL Auto cell imaging system, Thermo Fisher Scientific, USA). The number and ratio of live and dead cells were quantitatively analyzed using ImageJ software.

[0076] Live and dead cell staining showed that 3DP-HHO had high cell viability, comparable to that of hiHeps cultured in two dimensions. Figure 2 A, 2B). CCK-8 assay showed that the cell proliferation rate of 3DP-HHO was significantly higher than that of the two-dimensional culture system (A, B). Figure 2 C). After one month of cultivation ( Figure 2 D) 3DP-HHO shows hepatocyte spheroids (white arrows) and hepatic cord-like structures (red arrows), indicating that the organoid has matured.

[0077] Example 2: Analysis of Partition-Based 3D Bioprinting of Highly Expressed Hepatocyte Organoids Based on RNA Sequencing Cells within 3D printed hybrid liver organoids (3DP-HHOs) were lysed using GelMA lysis solution (Cat. No. EFL-GM-LS-001, EFL, China) for 1 hour at 37 °C in a carbon dioxide incubator. Subsequently, total RNA of 3DP-HHOs was extracted according to the instruction of RNA purification kit (Cat. No. AC0202, SBI, China) and the RNA concentration was detected using Nano Drop 2000 spectrophotometer.

[0078] RNA samples were prepared for high-throughput sequencing by Beijing Genescript Biotechnology Co., Ltd. Related RNA sequencing data has been approved and uploaded to the National Institutes of Health GEO database (Accession number: GSE298708; website: www.ncbi.nlm.nih.gov). Primary human hepatocytes and HepRG cell data were derived from published RNA-seq dataset (GEO: GSE205179, GPL24676), which contains data of primary human hepatocytes (PHH) and HepRG cells (n=3 biological replicates per group). Both GSE298708 and GSE205179 datasets were processed by a unified analysis pipeline. Gene expression levels were quantified as transcripts per kilobase fragment per million mapped reads (FPKM), followed by log2 transformation of FPKM values, with the formula log2(FPKM + 1). To correct for technical differences between the two datasets, the ComBat algorithm in the sva R package was used to remove batch effects. After removing batch effects, the data was standardized by Z-score and used for principal component analysis (PCA) and heat map plotting. This analysis was completed using the OmicStudio tool (https: / / www.omicstudio.cn / tool), and the results were visualized using the tools provided by the bioinformatics online platform (https: / / www.xiantaozi.com).

[0079] Transcriptome analysis showed that 3DP-HHOs had a significantly different gene expression profile from 2D cultured hiHeps, with key liver genes PCK1, PLIN2, SLC5A1 and SLC22A7 showing significant upregulation Figure 2 E). KEGG enrichment analysis revealed significant upregulation of drug metabolism, bile secretion and carbohydrate metabolism related pathways Figure 2 F). In addition, 3DP-HHOs showed a similar spatial partitioning pattern of marker genes to primary human hepatocytes (PHH), while this partitioning expression feature was not observed in 2D culture Figure 2 G).

[0080] OCT-embedded tissue sections or 3D-printed hybrid liver organoids (3DP-HHOs) were cryosectioned. Sections were thawed at room temperature for 30 minutes and then fixed with 4% paraformaldehyde for 30 minutes. Samples were permeabilized with enhanced immunostaining permeabilization buffer (catalog number: P0097-500 ml, Beyotime, China) at room temperature for 10 minutes, with PBS washing between each step. Subsequently, sections were blocked with 10% goat serum at room temperature for 1 hour, followed by overnight incubation at 4°C with primary antibody diluted in 10% goat serum. After washing with PBST, samples were incubated with fluorescently labeled secondary antibody at room temperature for 1 hour. Finally, nuclear counterstaining was performed using DAPI (catalog number: C1006-50 ml, Beyotime, China). All imaging was performed using a fluorescence microscope (EVOS FLAuto cell imaging system, Thermo Fisher Scientific, USA), and quantitative analysis was performed using ImageJ software. CYP3A4 is mainly expressed in the central hepatic cells near the central vein, while HAL is enriched in the porta hepatis cells near the portal vein. Figure 2 These data highlight the metabolic partitioning of the liver, which is crucial for maintaining its diverse physiological functions, including foreign body detoxification, amino acid metabolism, and nitrogen balance regulation. A similar partitioning pattern was also observed in the 3DP-HHO. Figure 2 I). Within self-assembled spherical liver organoids, HAL expression was highly concentrated in the central core region of the spheroid (marked by white pentagrams), while CYP3A4 expression was primarily confined to the peripheral periphery (marked by white arrows). This distribution likely reflects the spatial heterogeneity of oxygen and nutrient availability in the microenvironment, which regulates the metabolic partitioning of hepatocytes. Although similar to in vivo liver partitioning, the patterns observed in organoids may represent altered or reversed metabolic patterns driven by unique conditions in the in vitro system.

[0081] Example 3: Analysis of changes in liver function and metabolism in 3DP-HHO Transcriptome analysis showed that the gene expression profile of 3DP-HHO was more similar to that of primary human hepatocytes, especially in glucose, lipid and amino acid metabolism and urea cycle-related pathways. Figure 3 Based on the above findings, the robust and mature function of 3DP-HHO was further confirmed by the following histological staining experiments: PAS staining: hiHeps / 3DP-HHOs were fixed with 4% paraformaldehyde (Cat. No. FD3628-500, Fuheda, China) and then followed the protocol of PAS staining kit (Cat. No. G1360, Solarbio, China); Oil red O staining: hiHeps and 3DP-HHOs were treated with BSA control or OA (0.5 mM) and PA (0.25 mM) mixture (Cat. No. KC006, Kechuangkeji, China) for 48 hours, respectively, and then followed the protocol of oil red O staining kit (Cat. No. G1262, Solarbio, China). PAS staining showed glycogen storage, while OA+PA treatment highlighted lipid accumulation Figure 3 D, 3E) Quantitative functional assays further confirmed the liver function performance of 3DP-HHOs: protein concentration in culture supernatant or serum samples was determined by ELISA kit (Human Albumin Test Kit, RK00157, ABclonal, China). After appropriate dilution, samples were added to the microplate pre-coated with the target antigen along with a series of known standard concentration solutions, and all operations followed the product instructions. The absorbance value of each well was determined using a microplate reader, and the protein concentration was calculated by the standard curve.

[0082] Urea nitrogen concentration in culture supernatant was detected by urea nitrogen assay kit (C013-2-1, Nanjing Jiancheng Bioengineering Institute, China), and the operation strictly followed the protocol. The absorbance value was determined using a microplate reader, and the actual concentration was calculated according to the standard curve established by the urea nitrogen standard.

[0083] All staining and fluorescence imaging were completed using a Nikon brightfield microscope or fluorescence microscope (EVOS FL Auto Cell Imaging System, Thermo Fisher Scientific, USA). Compared with two-dimensional cultured hiHeps, 3DP-HHOs had significantly higher urea nitrogen production and albumin secretion, which reflected the liver-specific metabolic and synthetic functions Figure 3 F) In addition, the expression of drug metabolism-related genes (such as CYP enzymes) and bile secretion markers in 3DP-HHOs was significantly higher Figure 3 G, 3H) DiI-Ac-LDL uptake experiment further confirmed that 3DP-HHOs could internalize LDL Figure 3I), the specific experimental steps are as follows: hiHeps were co-incubated with 3DP-HHOs and 10 pg / mL Dil-Ac-LDL (Cat. No. 20606ES76, YEASEN, China) for 4 hours, followed by nuclear counterstaining using 1 pg / mL Hoechst 33342 (Cat. No. 40732ES03, YEASEN, China) for 15 minutes at 37°C. ICG uptake and elimination experiments further confirmed the hepatobiliary function of 3DP-HHOs, the specific experimental steps are as follows: hiHeps were exposed to 1 mg / mL ICG (Cat. No. HY-D0711, MCE, China) for 30 minutes at 37°C to complete the uptake. Subsequently, the samples were washed three times and transferred to ICG-free HMM medium for further incubation at 37°C, and the ICG release was observed and evaluated at 0, 2.5, 5, 10, and 24 hours after incubation, respectively. The results showed that, compared with 2D-cultured hiHep cells (which had very low ICG retention), 3DP-HHOs exhibited significant ICG uptake in a short time after exposure, and then gradually eliminated within 24 hours Figure 3 J). This dynamic ICG processing mode reflects the presence of active liver uptake and excretion mechanisms, highlighting the enhanced liver-specific functions of 3DP-HHOs. These results collectively highlight the enhanced functional maturity of 3DP-HHOs, demonstrating their potential as a more physiologically relevant in vitro model for studying liver function, metabolism, and drug response.

[0084] Example 4: Therapeutic effect of 3DP-HHOs on chronic liver fibrosis Chronic liver fibrosis, as a common cause of progressive liver failure, is usually driven by long-term liver damage induced by toxins such as thioacetamide (TAA) and carbon tetrachloride (CCl4). In some embodiments, a TAA-induced liver fibrosis mouse model was constructed Figure 4A), 8-week-old BALB / c nude mice (Hangzhou Suryuan Experimental Animal Technology Co., Ltd.) were used to establish the liver fibrosis model by intraperitoneal injection of thioacetamide (TAA, Cat. No. 60395ES25, Yixing, China). The dose was 200 mg / kg (dissolved in sterile PBS), and the injection was performed 3 times a week for 8 weeks. At the 6th week of modeling, the mice were anesthetized with isoflurane and a midline incision was made to open the abdomen. The 3D-printed hybrid liver organoids (3DP-HHO) were implanted into the left lateral lobe and the middle lobe of the mouse liver. The control groups included mice injected with saline, mice implanted with cell-free 3D-printed GelMA scaffolds, and mice injected with two-dimensional cultured human induced hepatocyte-like cells (hiHeps, 2.5 million cell suspension). After implantation, the abdominal wall was sutured in layers. The general condition, wound healing, and body weight changes of the mice were monitored daily after surgery. At the designated endpoint, the mice were euthanized, and serum and liver tissue samples were collected for histological staining, gene expression, and biochemical analysis to assess the degree of fibrosis.

[0085] In some embodiments, biochemical analysis was performed on the serum of the mice. Specifically, after the mice were anesthetized with isoflurane, about 300-500 μL of whole blood was collected through the retro-orbital plexus using a heparinized capillary tube. The blood sample was allowed to clot at room temperature for 30 minutes, and then centrifuged at 3000g for 10 minutes at 4°C. The supernatant serum was carefully transferred to a new centrifuge tube and stored at -80°C for testing. The fully automatic dry biochemical analyzer (DRI-CHEM NX700V, Fuji, Japan) was used for detection: 10 μL of thawed serum was added to the reagent sheet, and the enzyme activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB), alkaline phosphatase (ALP), total bilirubin (TBil), and γ-glutamyltransferase (GGT) were quantitatively detected according to the manufacturer's instructions.

[0086] The results are shown in Table 1. Figure 4B, 4C shows that the AST and ALT levels of the GelMA control group serum did not show significant improvement. The ALT of the hiHep cell transplantation group and the 3DP-HHO group was significantly reduced, indicating that liver function was enhanced. In some embodiments, histological analysis was performed on the stained liver tissue sections. All stained sections were imaged using a Nikon brightfield microscope and quantitatively analyzed by ImageJ software. Among them, the specific steps of HE staining are as follows: after the liver tissue is fixed with 4% paraformaldehyde overnight, it is sequentially dehydrated, transparentized and paraffin-embedded. The paraffin block is continuously sectioned, with a thickness of 5 μm. After standard dewaxing treatment, the liver sample is stained using a hematoxylin-eosin staining kit (item number: G1120, Soleris, China) to observe the histological morphology of the necrotic liver area; the specific experimental steps of Sirius red staining are as follows: after the tissue section is fixed, deparaffinized and hydrated, it is operated according to the instructions of the modified Sirius red staining kit (item number: G1472, Soleris, China); the specific experimental steps of Masson staining are as follows: after the tissue section is fixed, deparaffinized and hydrated, it is operated according to the instructions of the modified Masson staining kit (item number: G1346, Soleris, China). The results are shown in Figure 4 D shows that the TAA treatment group has significant fibrosis, and the transplantation of 3DP-HHO can significantly alleviate the fibrosis characteristics.

[0087] In some embodiments, a CCl4-induced liver fibrosis mouse model was constructed Figure 4 E), the difference between the two is that CCl4 (item number: C805325, Macklin, China) was injected into the abdominal cavity of the mouse, and the dose was 20 mL / kg. The serum analysis and tissue staining analysis are also referred to the above steps. The results are shown in Figure 4 F shows that after two weeks of transplantation, human serum albumin can be detected in the serum of the treatment group mice, and the protein level of the 3DP-HHO group is significantly higher than that of the hiHeps and GelMA control groups. Histological evaluation further shows that the liver fibrosis of the 3DP-HHO treatment group is significantly improved, showing restored parenchymal structure and reduced collagen deposition Figure 4G) In some embodiments, Western blot analysis is used to represent the changes at the molecular level. Specifically, the liver tissue samples are lysed with RIPA lysis buffer (Cat. No. FD009, Vazyme, China) with the addition of protease inhibitor cocktail (Cat. No. FD1001, Vazyme, China) and phosphatase inhibitor cocktail (Cat. No. FD1002, Vazyme, China) to extract proteins. The protein concentration is determined using the Pierce BCA Protein Assay Kit (Cat. No. A55865, Thermo Fisher Scientific, USA). After adjusting the protein concentration, the proteins are mixed with protein loading buffer (Cat. No. FD006, Vazyme, China) and denatured by boiling at 100°C for 10 minutes. Protein separation is performed using 4-20% FuturePAGE™ precast gels (Cat. No. ET15420L Gel, ACE, China), followed by transfer to PVDF membranes (Cat. No. IPVH00010, Millipore, USA). The membranes are blocked with Rapid Block (Cat. No. RM02956, Abclonal, China) for 15 minutes, then incubated with specific primary antibodies diluted at an appropriate ratio (1:1000 dilution of primary antibody diluent) overnight at 4°C. After TBST washing, the membranes are incubated with HRP-labeled secondary antibodies for 1 hour at room temperature. Finally, the protein signals are detected by enhanced chemiluminescence method, and the band gray values are analyzed using ImageJ software. The results are shown in Figure 4 H shows that the expression of a-SMA protein in the 3DP-HHO group is significantly down-regulated.

[0088] In some embodiments, the key fibrosis genes TGF-β, a-SMA, TIMP1, and COL1A1 are quantitatively analyzed by performing RT-qPCR on the sample RNA. Specifically, 1 pg of total RNA is used for cDNA synthesis by Hifair® II 1st Strand cDNA Synthesis Kit (Cat. No. 11119ES60, Yixing, China) after following the specific steps for RNA extraction in Example 1. cDNA quantification is performed using Hieff UNICON® qPCR SYBR Green Master Mix (Cat. No. 11200ES08, Yixing, China) on an Applied Biosystems QuantStudio qPCR system (Thermo, USA). In the experiment, b-actin / GAPDH is used as an internal reference. The results are shown in Figure 4 I shows that the expression of these genes in the 3DP-HHO implanted mice is significantly inhibited, which is consistent with the attenuation of fibrosis signaling. The results of the above examples collectively emphasize the potential of 3DP-HHO in promoting regeneration and anti-fibrosis, both restoring liver-specific functions and reducing pathological remodeling in the late fibrosis environment.

[0089] Example 5: Therapeutic effect of 3DP-HHO on acute liver failure In some embodiments, an APAP-induced acute liver failure model is constructed Figure 5 A), with the specific steps referring to the mouse model construction in Example 4, except that the mice are injected intraperitoneally with APAP (Cat. No. 53626ES08, Yisen, China) at a dose of 300 mg / kg (dissolved in sterile PBS), and the fine implantation of 3DP-HHOs is performed 24 hours after modeling. The control group is mice injected intraperitoneally with normal saline. The mice in each group are sacrificed at 24, 48, and 72 hours after APAP injury, respectively, to evaluate liver damage, necrosis, and regeneration. Serum analysis and tissue staining analysis also refer to the steps in Example 4. The results are shown in Figure 5 B, the serum ALT level as a marker of liver cell injury reaches a peak at 24 hours, and all groups show a decreasing trend at 48 hours. The implantation of 3DP-HHO significantly accelerates the normalization of ALT level, and shows a dose-dependent effect, with the 2.5M and 5M groups showing the greatest reduction in ALT compared to the control group. Liver tissue HE staining shows that extensive necrosis occurs in all groups at 24 hours, and the necrotic area is significantly reduced in the 3DP-HHO treatment groups (especially the high-dose group) at 48 hours Figure 5 C). Figure 5 F).

[0090] In some embodiments, the proliferation of liver cells is detected by Ki67 immunohistochemistry. The specific steps are as follows: the section is placed in 0.01 M sodium citrate buffer and heated at 95°C for 20 minutes for antigen retrieval, and then the operation is performed according to the instructions of the immunohistochemistry kit (Cat. No. 36311ES50, Yisen, China). The primary antibody used is Ki67 Rabbit PolymAb®(Cat. No. A26419-PM, ABclonal, China), and the primary antibody diluent is diluted at 1:1000. The results are shown in Figure 5 D, 5G, 72 hours after APAP injury, the number of Ki67-positive liver cells in the 3DP-HHO group is significantly increased compared to the control group, indicating enhanced cell proliferation. Western blot analysis further explores the molecular mechanism of liver cell proliferation. As shown in Figure 5 E, 5H, the cell cycle regulatory factors have changed significantly: CDK4 is significantly down-regulated, while CDK1 is up-regulated. The liver cells in the 3DP-HHO group may have entered the G2 / M phase from the G1 phase with active CDK4, which may be driven by the activation of CDK1. This transition indicates that 3DP-HHO may accelerate the cell cycle transition and promote the timely entry of cells into mitosis during liver regeneration.

[0091] In some embodiments, a CCl4-induced acute liver failure (ALF) mouse model was constructed. Figure 6 A) The specific steps are the same as those described above for constructing the APAP-induced acute liver failure model, with the difference being: mice were intraperitoneally injected with CCl4 at a dose of 20 mL / kg (CCl4 diluted to a 10% concentration using sterile corn oil). Mice in each group were sacrificed at 24, 48, and 72 hours after CCl4 injury to assess liver damage, necrosis, and regeneration. Serum analysis and tissue staining analysis were performed according to the steps in Example 4. Results are as follows... Figure 6 As shown in Figure B, serum ALT levels, a marker of hepatocellular injury, peaked at 24 hours and then gradually decreased in all groups up to 48 hours. Implantation of 3DP-HHO significantly accelerated the normalization of ALT levels in a dose-dependent manner, with the 5M group showing the most significant reduction compared to the control group. Figure 6 B). Histopathological examination by HE staining showed that at both 48 and 72 hours, the structural integrity of the livers of mice treated with 3DP-HHO was preserved, and the degree of necrosis was significantly reduced compared to the control group. In some embodiments, hepatocyte death was further assessed by TUNEL staining. The specific steps were as follows: frozen sections of OCT-embedded tissue samples were prepared. The sections were warmed to room temperature for 30 minutes, then fixed with 4% paraformaldehyde for 30 minutes, and then stained according to the instructions of the TUNEL apoptosis detection kit (catalog number: C1088, Beyotime, China). Finally, the samples were imaged and observed using a fluorescence microscope (EVOS FL Auto cell imaging system, Thermo Fisher Scientific, USA). The results are as follows. Figure 6 As shown in Figure C, 48-hour TUNEL staining revealed a large number of apoptotic cells in the control group, while 3DP-HHO treatment significantly reduced TUNEL-positive cell nuclei, indicating that 3DP-HHO treatment effectively alleviated CCl4-induced hepatocyte apoptosis.

[0092] In some embodiments, the results of Ki67 immunohistochemical detection of hepatocyte proliferation showed ( Figure 6 D), 72 hours later, the number of Ki67-positive cell nuclei in the 3DP-HHO group was significantly higher than that in the experimental group, indicating enhanced hepatocyte proliferation. Western blot analysis of cell cycle-related markers showed that PCNA, CDK1, and pH3S10 were significantly upregulated in the 3DP-HHO group, indicating active cell cycle progression and entry into the mitotic stage. Figure 6 E).

[0093] Example 6: Effect and mechanism of 3DP-HHO on the proliferation of hepatocytes in a partially hepatectomized mouse model In some embodiments, a 2 / 3 (70%), 85%, 90% partial hepatectomy (PHx) mouse model is constructed, and the specific steps are as follows: 8-week-old BALB / c nude mice are anesthetized with isoflurane and placed on a thermostatic surgery platform. A midline incision is made to expose the liver: standard 2 / 3 hepatectomy model (70% PHx): according to the method in the literature “A reproducible and well-tolerated method for 2 / 3 partial hepatectomy in mice”, the left lateral lobe and the middle lobe are ligated and resected; standard 85% hepatectomy model: according to the method in the literature “Reversal of liver failure using a bioartificial liver device implanted with clinical-grade human-induced hepatocytes”, the left lateral lobe, the middle lobe and the right upper lobe are ligated and resected, only the caudate lobe and the right lower lobe are reserved; 90% hepatectomy model: according to the method in the literature “C-C motif chemokine ligand 5 confines liver regeneration by down-regulating reparative macrophage-derived hepatocyte growth factor in a forkhead box O 3a–dependent manner”, additional resection of the right lower lobe is performed based on the 85% resection. After the above models are resected, the 3DP-HHO is implanted into different anatomical sites according to the liver resection range: the 70% PHx model is implanted between the right upper lobe and the right lower lobe; the 85% PHx model is implanted on the surface of the right lower lobe; the 90% PHx model: implanted on the surface of the caudate lobe. After implantation, the abdominal cavity is flushed with warm sterile saline, and the abdominal wall is sutured in two layers. The survival, wound healing and overall clinical condition of the animals are observed daily. According to the relevant methods in the above embodiments 1-5, liver tissue and serum samples are collected at specific time points, and liver regeneration is evaluated by histology, transcriptome and protein levels.

[0094] In some embodiments, the results of the construction of the 2 / 3 partial hepatectomy (PHx) model (also referred to as the 70% PHx model) are as shown in FIG. A, and samples are collected at 24, 48 and 96 hours after the PHx surgery in mice. Compared with the control group, the mice receiving 3DP-HHO have a significantly higher liver-to-body weight ratio at 48 hours, indicating accelerated liver mass recovery Figure 7 A). At the same time, the serum ALT and AST levels of the 3DP-HHO group are lower, indicating reduced surgical stress or improved metabolic recovery Figure 7 B). At the same time, the serum ALT and AST levels of the 3DP-HHO group are lower, indicating reduced surgical stress or improved metabolic recovery Figure 7C).

[0095] In some embodiments, histological analysis showed that the 3DP-HHO group retained lobular structure and increased cell density after 48 hours, accompanied by a significant increase in Ki67-positive hepatocytes. This result was verified by immunohistochemical staining and quantitative analysis. Figure 7 D, 7E). Western blot analysis ( Figure 7 (F, 7H) showed that PCNA, CDK1, and phosphorylated histone H3 (pH3S10) were significantly upregulated in the 3DP-HHO group. qRT-PCR showed that, compared with the control group, liver treated with 3DP-HHO showed significantly upregulated expression of Cyclin D1, B1, A2, and E1 at 48 and 96 hours. Figure 7 G) indicates that the cell cycle transcription program has been extensively activated. These results strongly support the role of 3DP-HHO in promoting regeneration after hepatectomy.

[0096] Hepatocyte growth factor (HGF) is primarily activated through MMP-mediated cleavage and exerts a key mitotic effect by binding to c-Met receptors on the surface of hepatocytes. The temporal coordination between MMPs and HGF ensures a microenvironment conducive to liver tissue repair and the necessary mitotic stimulation.

[0097] In some embodiments, gene expression analysis showed that MMP2 and MMP9 were significantly upregulated in hiHeps cultured with 3DP-HHO compared to the two-dimensional culture group. Figure 8 A) Matrix metalloproteinases (MMPs) promote the proliferation and migration of hepatocytes during regeneration by regulating extracellular matrix remodeling, indicating enhanced extracellular matrix remodeling capacity. The ELISA analysis was performed following the specific steps in Example 2, with the following differences: human MMP-2 detection kit (RK00309, ABclonal, China), human MMP-9 detection kit (RK00217, ABclonal, China), and mouse HGF detection kit (RK00371, ABclonal, China) were used for detection. The results showed that the 3DP-HHO group had significantly higher MMP2 release (…). Figure 8 B). In the 70% PHx model, serum HGF levels in the 3DP-HHO group were significantly higher than those in the sham-operated group at 24 hours post-operation. Figure 8 (C) Hepatocyte growth factor (HGF) is mainly activated by MMP-mediated cleavage and plays a key role in promoting mitosis by binding to c-Met receptors on the surface of hepatocytes, indicating enhanced regenerative signaling. Histological analysis confirmed that livers treated with 3DP-HHO exhibited enhanced cell proliferation and tissue remodeling, manifested by a significant increase in Cyclin D1 expression levels. Figure 8F, 8H). HGF can induce the expression of Cyclin D1, a key factor that regulates the transition from G1 to S phase. This upregulation of Cyclin D1 supports the accelerated DNA synthesis and enhanced cell proliferation observed in the regenerating liver tissue.

[0098] In some embodiments, Transwell migration experiments of hiHeps were performed, with the specific steps as follows: 24-well Transwell chambers with polycarbonate membranes of 8 pm pore size (Corning) were used to assess cell migration capacity, hiHeps cultured in 2D or 3DP-HHO were seeded in the upper chamber. The lower chamber was filled with HMM. After incubation at 37 °C for 24 or 48 h, the non-migrated cells on the upper chamber surface were removed with a cotton swab. The migrated cells on the lower chamber surface were fixed with 4% paraformaldehyde, stained with crystal violet, and imaged under a light microscope. The stained cells in five randomly selected fields of each well were counted using ImageJ software, thereby quantifying cell migration.

[0099] The results showed that the migration capacity of hiHeps cultured in 3DP-HHO matrix was significantly higher than that of the 2D culture group Figure 8 D), and the difference was quantitatively analyzed by the number of migrated cells per field Figure 8 G).

[0100] In some embodiments, the survival rate of mice receiving 3DP-HHO treatment was significantly improved in 85% and 90% liver resection models Figure 8 I-J), demonstrating that enhancing liver regeneration capacity is of great significance to prolong life. Figure 8 K summarizes the pathways by which 3DP-HHO promotes proliferation and regeneration, and these findings collectively emphasize the powerful regenerative and survival benefits conferred by 3DP-HHO in severe liver injury models.

[0101] Example 7: Therapeutic effect of 3DP-HHO on a pig model of liver failure after liver resection In some embodiments, a porcine partial hepatectomy followed by liver failure (PHLF) model was constructed. The specific steps were as follows: Following the method described in the literature "Reversal of liver failure using a bioartificial liver device implanted with clinical-grade human-induced hepatocytes," extended (85%) hepatectomy was performed on Bama miniature pigs (Wujiang Tianyu Biotechnology Co., Ltd., SCXK(Su)2021-0007). A simplified procedure was as follows: Female pigs (weighing 20-25 kg, 4-6 months old) were fasted for 24 hours preoperatively. After general anesthesia, a right subcostal incision was made to expose the liver. The left lateral lobe, left medial lobe, right medial lobe, and most of the right lateral lobe were surgically removed, leaving only the caudate lobe intact. Major hepatic vessels and bile ducts were carefully separated, ligated, and severed using electrosurgical instruments and an ultrasonic scalpel. Postoperatively, analgesia and supportive care were administered according to standard protocols, including intravenous fluid resuscitation, antibiotics, and glucose infusion. The pigs were housed separately and their recovery was observed. Water was withheld for 6 hours postoperatively, and food was withheld for 24 hours. After observing the pigs defecating, initially feed them a liquid diet, gradually transitioning to a semi-liquid diet, and finally resuming normal feeding. Monitor the pigs' survival, wound healing, and overall clinical condition daily. Collect liver tissue and serum samples at specific time points. After anesthetizing the pigs, collect approximately 3 mL of whole blood from the jugular vein using a disposable lancet. Subsequent steps are as described in the serum analysis in Example 4.

[0102] High-density 3D-printed 3DP-HHO was immobilized on the surface of residual liver tissue for treatment, following the method described in the literature "Reversal of liver failure using a bioartificial liver device implanted with clinical-grade human-induced hepatocytes". The control group received no treatment. Results showed that pigs not treated with 3DP-HHO exhibited lethargy and liver failure due to insufficient residual liver volume, dying within 24-36 hours post-surgery. Pigs in the 3DP-HHO treatment group showed significantly improved activity levels. Figure 9 A), median survival increased from 30 hours to 120 hours. Figure 9 B). One week later, these pigs returned to normal vitality. Continuous volume assessment showed that the residual liver volume in the 3DP-HHO treatment group increased significantly within 24 hours post-surgery, indicating accelerated liver regeneration. Figure 9 C). Postoperative CT results of the porcine PHLF model showed that the residual liver volume was approximately 14.37%. One week after surgery, the residual liver weight in the treatment group reached 74.99% of the average liver weight of healthy Bama miniature pigs. Figure 9D) Histological analysis further confirmed the regeneration process: HE staining of the residual liver tissue at 24 h, 36 h, 72 h and 168 h post-operation showed that 3DP-HHOs were closely attached to the liver surface, with similar morphology to the native liver tissue Figure 9 E) The implanted 3DP-HHOs were marked with yellow pentagrams, and the pig liver tissue was marked with white pentagrams.

[0103] In some embodiments, serum biochemical analysis showed that the levels of total bilirubin (TBil), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) in the 3DP-HHO group were significantly decreased at 24 h after treatment Figure 9 F) In the HE staining analysis at 24 h after treatment, the liver lobule size in the 3DP-HHO group was significantly larger than that in the control group Figure 9 G), indicating that the regeneration activity was enhanced. In addition, Ki67 immunohistochemical staining showed that the proportion of proliferative hepatocytes in the liver of the treatment group was significantly increased, and quantitative evaluation confirmed that the Ki67 positive cell index was significantly up-regulated Figure 9 G), which jointly confirmed that the therapy had the ability to stimulate cell proliferation at an early stage. In summary, the 3DP-HHO implant can effectively promote the regeneration of the residual liver, and the liver mass can be restored to about 85% after extensive resection. This regeneration response is crucial for preventing liver failure, highlighting that bio-printed liver organoids not only restore liver function, but also accelerate tissue regeneration, thus providing a promising strategy for reducing the risk of major liver surgery.

[0104] The above description of the embodiments is only for the purpose of understanding the method of the present application and its core idea. It should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and modifications can be made to the present application, and these improvements and modifications will also fall within the scope of protection of the claims of the present application.

Claims

1. A method for 3D printing liver organoids, characterized in that, The liver organoid was obtained by 3D printing using cell bio-ink; Preferably, the cell bio-ink includes hiHep cells and pre-made bio-ink.

2. The method according to claim 1, characterized in that, The pre-made bio-ink includes natural polymer materials, synthetic polymer materials, composite hydrogel materials, and extracellular matrix-derived materials; Preferably, the bio-ink is a natural polymer material; Preferably, the natural polymeric materials include GelMA, gelatin, collagen, hyaluronic acid, sodium alginate, silk protein, and fibroin; Preferably, the natural polymer material is GelMA; Preferably, the concentration of GelMA is 3% to 10%; Preferably, the concentration of GelMA is 5%; Preferably, the pre-made bio-ink further includes a crosslinking initiator; Preferably, the crosslinking initiator includes a photocrosslinking initiator, an ionic crosslinking initiator, an enzymatic crosslinking initiator, and a thermosensitive crosslinking initiator; Preferably, the crosslinking initiator is a photocrosslinking initiator; Preferably, the photocrosslinking initiator is LAP; Preferably, the concentration of LAP is 0.05% to 0.5%; Preferably, the concentration of LAP is 0.25%; Preferably, the pre-made bio-ink further includes a hepatocyte maintenance culture medium.

3. The method according to claim 1, characterized in that, The 3D printing specifically includes the following operations: placing the cell bio-ink as described in claim 1 or 2 into the nozzle of a 3D printing device and performing layer-by-layer extrusion printing; Preferably, the nozzle includes an extrusion nozzle, a jet nozzle, or a UV-curing nozzle; Preferably, the extrusion nozzle includes a pneumatically driven nozzle, a screw-driven nozzle, and a piston-driven nozzle; Preferably, the extrusion nozzle is a pneumatically driven nozzle; Preferably, the pneumatically driven nozzle includes a syringe barrel, a needle, a drive module, and a temperature control module; Preferably, the needle has a weight of 27G to 33G; Preferably, the needle is 32G; Preferably, the temperature control module is set to a temperature of 10℃~20℃; Preferably, the temperature control module is set to 11°C.

4. The method according to claim 1, characterized in that, The method also includes photocrosslinking curing and culture, specifically including the following operations: the printed structure is irradiated with a light source to achieve photocrosslinking curing for 30-60 seconds, and the crosslinked structure is transferred to hepatocyte maintenance culture medium, with the culture medium being changed every two days; Preferably, the light source is ultraviolet light, visible blue light, or near-ultraviolet long-wave light; Preferably, the light source is visible blue light; Preferably, the wavelength of the visible blue light is 405-480 nm; Preferably, the wavelength of the visible blue light is 405 nm.

5. A raw material for printing liver organoids, characterized in that, The raw materials include hiHep cells and / or pre-made bio-ink as described in any one of claims 1-3; Preferably, the hiHep cells are harvested from hepatocyte maintenance culture medium; Preferably, the hepatocyte maintenance culture medium comprises a basal culture medium and functional additives; Preferably, the basal culture medium includes DMEM / F12 and 10% fetal bovine serum; Preferably, the functional additives include growth factors, hormones, micronutrient supplements, nerve cell supplements, and maintenance additives; Preferably, the hepatocyte maintenance culture medium comprises any one of insulin-transferrin-selenium, SM1 nerve cell supplement, TGF-α, EGF, or dexamethasone; Preferably, the culture medium consists of insulin-transferrin-selenium, SM1 nerve cell supplement, 40 ng / mL TGF-α, 40 ng / mL EGF, and 10 μM dexamethasone. Preferably, the pre-made bio-ink includes GelMA and LAP; Preferably, the concentration of GelMA is 3% to 10%; Preferably, the concentration of the GelMA solution is 5%; Preferably, the concentration of LAP is 0.05% to 0.5%; Preferably, the concentration of LAP is 0.25%; Preferably, the pre-made bio-ink further includes a hepatocyte maintenance culture medium; Preferably, the hiHep cells are suspended in bio-ink at a certain cell density; Preferably, the cell density is 1×10⁻⁶. 7 Or 5×10 7 per mL.

6. A liver organoid or tissue, characterized in that, The liver organoid or tissue is prepared by the method described in any one of claims 1-4.

7. The application of the method according to any one of claims 1-4, the raw material according to claim 5, and the liver organoid or tissue according to claim 6 in 3D printing liver models; Preferably, the liver-like model is a liver-like primary lobule partitioning structure model.

8. The use of the method according to any one of claims 1-4, the raw material according to claim 5, and the liver organoid or tissue according to claim 6 in evaluating the efficacy of a medicament for treating liver-related diseases; Preferably, the disease includes one or more of the following: acute liver injury, drug-induced liver injury, acute or chronic liver failure, liver fibrosis, cirrhosis, non-alcoholic steatohepatitis, hereditary metabolic liver disease, cholestatic hepatitis, or hepatocellular carcinoma.

9. The application of the method according to any one of claims 1-4, the raw material according to claim 5, and the liver organoid or tissue according to claim 6 in constructing an in vitro liver function reconstruction system; Preferably, the liver function includes protein synthesis function, ammonia metabolism function, drug metabolism function, bilirubin metabolism function, energy metabolism function, and toxicity response function.

10. The application of the method according to any one of claims 1-4, the raw material according to claim 5, and the liver organoid or tissue according to claim 6 in constructing a bioartificial liver system; Preferably, the bioartificial liver system includes a bioreactor module, an extracorporeal circulation module, and an auxiliary module.

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