Organ-like-based functionalized biological 3D printing liver and construction method and application thereof

Abstract

The invention discloses an organoid-based functionalized biological 3D printing liver as well as a construction method and application of the organoid-based functionalized biological 3D printing liver. Liver organoid is used as seed cells, specific composite bio-ink is matched, liver tissue is constructed through a biological 3D printing technology, and the obtained construct is close to a natural liver in structure and function and shows high functional maturity including glycogen storage, albumin secretion and the like. The construction body can show the blood vessel integration capacity in animal transplantation experiments, and a new solution is provided for hepatic failure and drug toxicology screening.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically to a functionalized bio-3D printed liver based on organoids, its construction method, and its application. Background Technology

[0002] The liver is the body's main metabolic and detoxification organ, undertaking key functions such as glycogen storage, drug metabolism, plasma protein synthesis, and bile secretion. Liver failure and end-stage liver disease seriously threaten human life and health. Clinically, liver transplantation is the main treatment method, but due to issues such as donor shortage, immune rejection, and surgical risks, clinical needs are far from being met.

[0003] In recent years, the development of bioartificial livers and 3D bioprinting technology has provided new ideas for the in vitro construction of liver tissue. For example, CN116872491A discloses a 3D-printed biomimetic vascularized liver model and its printing method. This invention develops a block-shaped gel support medium called OPEN, combines this medium with compatible bio-ink, and creates a vascularized liver model that maintains liver function in vitro and promotes angiogenesis in vivo. CN114381419A relates to a biomimetic artificial liver tissue and its preparation method and application. This technology prepares liver tissue structures containing microfilaments and hollow channels through molding or 3D printing processes, with a cell density as high as 10-1. 7 ~10 8 It has cells / mL and possesses key functions such as albumin secretion, drug metabolism, nitrogen metabolism, and urea synthesis.

[0004] While the aforementioned bioprinting liver tissue technologies have made progress in vascularization and high cell density, driving the development of biomimetic liver tissue, they all rely on directly printing hepatocytes. Therefore, the following shortcomings remain: when directly using hepatocytes for printing, the cells are prone to dedifferentiation, and their function gradually declines, making it difficult to maintain stable liver function in the long term; existing technologies still have limitations in functional maturity. The emerging liver organoid technology can form three-dimensional structures in vitro through self-organization, maintaining the phenotype and function of hepatocytes better, and outperforming conventional two-dimensional cultured hepatocytes in areas such as albumin secretion. However, the volume and scale of organoids are limited, making direct application to clinical transplantation or large-volume tissue construction difficult. Current technologies have not effectively combined the functional maturity advantages of organoids with the scalability and structural controllability of 3D bioprinting. Therefore, there are still technological gaps to be addressed in the bioprinting of liver tissue that balance functional maturity and structural scalability. Summary of the Invention

[0005] To address the aforementioned issues, this invention provides a method for constructing a functionalized bioprinted liver based on organoids. Combining the functional advantages of organoids with the structural and scalable potential of bioprinting, and through innovative bio-inks and printing strategies, it achieves the in vitro construction of vascularized, highly functionally mature, and large-scale liver tissue.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for constructing a functionalized bio-3D printed liver based on organoids includes the following steps:

[0008] (1) Primary hepatocytes were induced in vitro to obtain liver organoid suspension;

[0009] (2) Preparation of composite bio-ink;

[0010] (3) The liver organoid suspension was mixed with the composite bio-ink and the mixture was transferred to a 3D bioprinter for printing to obtain a pre-gelled mesh;

[0011] (4) Cross-link the pregel mesh at room temperature and then perform functional maturation culture to obtain a functionalized bio-3D printed liver.

[0012] Preferably, the liver organoid suspension described in step (1) is prepared by the following method: primary hepatocytes are digested into single cells, and then subjected to a reaction at a ratio of 5 × 10⁻⁶ cells / mL. 6 The cells were seeded into the culture medium at a density of cells / mL and placed in hydrophobic culture dishes for 2 days at 37°C, 5% carbon dioxide, 90% humidity, and 100 rpm.

[0013] Preferably, the primary hepatocytes mentioned above are derived from humans or mice.

[0014] Preferably, the culture medium comprises: DMEM / F121X, ZnCl2 0.6 mg / L, ZnSO4·7H2O 1 mg / L, MnSO4 0.1 mg / L, Bovine serum albumin 5 g / L, Galactose 4 g / L, Ornithine 0.5 g / L, Proline 0.08 g / L, Nicotinamide 0.1 g / L, TGF 40 ng / ml, EGF 40 ng / ml, Dexamethasone 10 μM, and Fetalbovine serum 1%.

[0015] Preferably, the composite bio-ink in step (2) consists of: 10% (w / v) gelatin, 2% (w / v) sodium alginate, 8% (w / v) methacrylamide gelatin and 2% (w / v) hyaluronic acid.

[0016] Preferably, the gelatin is derived from commercially available cell culture-grade porcine skin gelatin, with a 300 Bloom, providing suitable gel strength, a moderate molecular weight range, and wide applicability in tissue engineering;

[0017] Preferably, sodium alginate has a molecular weight of 100-300 kDa, which ensures both printability and stability in Ca2+ environments. 2+ After cross-linking, a suitable mechanical strength is formed, and the molecular weight of sodium alginate is more preferably 200 kDa;

[0018] Preferably, the grafting rate of methacrylamide gelatin is 55-65%, which balances cross-linking efficiency and cell compatibility;

[0019] Preferably, the molecular weight of hyaluronic acid is 500-800 kDa, which is closer to the viscoelasticity of tissue ECM and is beneficial for simulating the matrix.

[0020] Preferably, the mixing in step (3) is as follows: the concentration of the liver organoid suspension is 1×10⁻⁶. 7 When the cell / mL ratio is 1, the volume ratio of the cell / mL ...

[0021] Preferably, step (3) of transferring the mixture to the 3D bioprinter for printing includes the following operations: transferring the mixture to the 3D bioprinter, placing the printing syringe at 4°C, and printing with the nozzle when the mixture turns into a gel to obtain a pre-gelled mesh.

[0022] Preferably, the parameters of the 3D bioprinter are set as follows: platform temperature 8℃, printing syringe temperature 15℃, printing pressure 40kPa, printing speed 5mm / s; the printing scanning spacing is 1500μm, single-track width is 500μm, and layer thickness is 300μm.

[0023] Preferably, step (4) includes the following steps:

[0024] (a) Crosslink the pregel mesh with 2.5% (w / v) CaCl2 solution at room temperature for 5 min, remove the residual CaCl2 solution, and obtain the 3D printed construct;

[0025] (b) The printed construct was placed in hepatocyte maturation medium and cultured at 37°C and 5% CO2 for 7 days, with the medium being changed every two days during the period.

[0026] Preferably, the hepatocyte maturation culture medium consists of: DMEM / F121X, ZnCl2 0.6 mg / L, ZnSO4·7H2O 1 mg / L, MnSO4 0.1 mg / L, Bovine serum albumin 5 g / L, Galactose 4 g / L, Ornithine 0.5 g / L, Proline 0.08 g / L, Nicotinamide 0.1 g / L, TGF 40 ng / ml, EGF 40 ng / ml, Dexamethasone 10 μM, and Fetal bovine serum 1%.

[0027] Another object of the present invention is to provide a functionalized bio-3D printed liver, which is obtained by the above-described construction method.

[0028] Another object of the present invention is to provide any of the following applications of the above-described functionalized bio-3D printed liver:

[0029] 1) Preclinical drug testing;

[0030] 2) Materials used in regenerative medicine or in vivo transplantation;

[0031] 3) Used for research on bioartificial livers or liver function compensation;

[0032] 4) Pathological research on liver diseases;

[0033] 5) New drug research and development.

[0034] As described above, this invention proposes a method for constructing a functionalized bio-3D printed liver based on organoids. Using liver organoids as seed cells and a specific composite bio-ink, liver tissue is constructed through bio-3D printing technology. The resulting functionalized bio-3D printed liver closely resembles the natural liver in both structure and function, exhibiting high functional maturity (such as glycogen storage and albumin secretion). This method overcomes the traditional limitations of organoid volume, achieving large-scale, structurally controllable liver tissue construction while retaining the self-organizing ability and functional advantages of organoids, thus achieving a unified technical effect of functional maturity and structural precision. Attached Figure Description

[0035] Figure 1 Microscopic observation of ICG uptake-release experiment in liver organoids (scale bar 100 μm), A: uptake, B: release;

[0036] Figure 2 Microscopic illustration of a 3D printed construct (scale bar 500μm);

[0037] Figure 3: Results of ALB (albumin) expression staining experiment in functionalized bioprinted liver (scale bar 100μm);

[0038] Figure 4 PAS staining results of functionalized bioprinted liver (scale bar 100μm);

[0039] Figure 5 Microscopic observation of ICG uptake-release experiment in functionalized bioprinted liver (scale bar 100μm), A: uptake, B: release;

[0040] Figure 6 Image showing the vascular characterization results of a functionalized bioprinted liver after in vivo transplantation (scale bar 500μm);

[0041] Figure 7 Comparison of liver function gene expression in 3D-printed livers obtained from different seed cells. The vertical axis represents the relative fold increase of each group relative to 3D-printed livers based on hepatocytes (normalized to 1). A: Cyp2b10 expression, B: Ttr expression.

[0042] Figure 8 Comparison of liver function gene expression in bio-3D printed livers prepared with bio-inks of different compositions. The vertical axis represents the relative multiple of each group relative to the composite bio-ink (normalized to 1); A: Alb expression, B: Ttr expression. Detailed Implementation

[0043] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.

[0044] All reagents used in the embodiments of this invention were purchased from commercial channels. Methods not mentioned are conventional experimental methods and will not be described in detail here.

[0045] The following reagents are provided as examples:

[0046] Gelatin (CAS: 9000-70-8) was purchased from Sigma-Aldrich, item number: V900863, purity grade: reagent grade.

[0047] Sodium alginate (CAS: 9005-38-3) was purchased from Sigma-Aldrich, item number: 180947.

[0048] Methacrylamide gelatin was purchased from Shangpu Boyuan (Beijing) Biotechnology Co., Ltd.

[0049] Hyaluronic acid (CAS: 9004-61-9) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., item number: 9004-61-9.

[0050] Example 1: Construction and Performance Testing of Liver Organoids

[0051] The following are the specific steps for rapidly constructing liver organoids from primary mouse hepatocytes by horizontal shaking:

[0052] Cultured mouse hepatocytes were digested into single cells and seeded at a density of 5 million cells per milliliter into a culture medium (composition: DMEM / F121X, ZnCl2 0.6 mg / L, ZnSO4·7H2O 1 mg / L, MnSO4 0.1 mg / L, Bovine serum albumin 5 g / L, Galactose 4 g / L, Ornithine 0.5 g / L, Proline 0.08 g / L, Nicotinamide 0.1 g / L, TGF 40 ng / ml, EGF 40 ng / ml, Dexamethasone 10 μM, Fetal bovine serum 1%). The cells were placed in hydrophobic culture dishes and then transferred to a shaker in an incubator at 37°C, 5% CO2, 90% humidity, and 100 rpm. On the second day of culture, the organoids had clumped together and were of uniform size. The primary mouse hepatocytes were isolated using a two-step collagenase perfusion method, specifically: the entire process was performed under aseptic conditions; mice were anesthetized and fixed on the operating table; the abdomen was opened to expose the liver and portal vein; and calcium-free collagenase was perfused at a constant temperature (37℃) of 7 mL / min. 2+ EBSS (60 mL) until the liver turns pale; switch to a solution containing Ca. 2+ Collagenase IV (0.05% w / v) dissolved in EBSS was perfused into 150 mL (37℃ constant temperature, 7 mL / min flow rate). After perfusion, the liver was aseptically removed, and enzyme activity was terminated using DMEM. The tissue was minced and the cells were released by pipetting. The cell suspension was filtered through a 100 μm cell sieve, centrifuged at 180 g × 5 min, the supernatant was discarded, and the pellet was retained. Percoll gradient enrichment was performed as needed (cells were resuspended in Percoll mixture, centrifuged at 180 g × 5 min, and the pellet was collected), followed by washing twice with PBS. The cells were then subjected to a 1 × 10⁻⁶ ppm solution. 4 cells / cm 2The cells were seeded at a density of type I collagen-coated culture dishes and adhered to the culture medium in DMEM containing 10% FBS for 6 hours. Then, the medium was replaced with hepatocyte expansion medium (composition: DMEM / F121X, ZnCl2 0.6 mg / L, ZnSO4·7H2O 1 mg / L, MnSO4 0.1 mg / L, Bovine serum albumin 5 g / L, Galactose 4 g / L, Ornithine 0.5 g / L, Proline 0.08 g / L, Nicotinamide 0.1 g / L, TGF 40 ng / ml, EGF 40 ng / ml, Dexamethasone 10 μM, Fetalbovine serum 1%) to support expansion and functional maintenance.

[0053] The ICG uptake and release capacity of the above organoids was tested using indocyanine green ICG (catalog number I2633-50MG, brand Sigma). The specific method was as follows: Hepatocyte expansion medium was discarded, and 1 mg / mL of ICG working solution (diluted with hepatocyte expansion medium) was added. After incubation at 37°C and 5% CO2 for 60 min, the cells were washed twice with PBS and immediately observed under a microscope. The results showed clearly visible green fluorescence (see...). Figure 1 A, uptake); replace with fresh culture medium (without IGG), continue incubation at 37℃ and 5% CO2 for 6 hours, and fluorescence was significantly reduced under a microscope (see...). Figure 1 B, release), indicates that the organoids exhibit ICG uptake and release capabilities, and can serve as seed cells for 3D-printed livers.

[0054] Example 2: Construction of a 3D-printed liver based on liver organoids

[0055] Preparation of composite bio-ink: (1) Preparation of mother liquor: At a ratio of 32% w / v, add methacrylamide gelatin powder to ddH2O preheated to 45-50℃, stir magnetically until completely dissolved, and filter sterilely through a 0.22μm filter membrane to obtain a 32% w / v GelMA mother liquor; At a ratio of 40% w / v, slowly sprinkle gelatin into ddH2O at 40-45℃ and stir to dissolve, and filter warmly through a 0.22μm filter to obtain a 40% w / v gelatin mother liquor; At a ratio of 8% w / v, add sodium alginate into ddH2O at 37℃ and stir magnetically to dissolve, and filter sterilize through a 0.22μm filter to obtain an 8% w / v sodium alginate mother liquor; At a ratio of 8% w / v, gradually sprinkle hyaluronic acid into ddH2O at room temperature, stir slowly for several hours until the solution is uniform and transparent, and filter sterilize through a 0.22μm filter to obtain an 8% w / v HA mother liquor. After all the mother liquors are prepared, keep them at 37°C. (2) Mixing: At 37°C, mix the four mother liquors of the above in equal volumes (1:1:1:1). The mixing order is as follows: first mix gelatin and sodium alginate, then add GelMA, and finally add HA. If microbubbles appear during the process, centrifuge briefly (at low speed, 4°C) to remove the bubbles, mix well, and you will get the composite bio-ink with the target final concentration.

[0056] The liver organoid suspension obtained in Example 1 (containing approximately 1 × 10⁶ cells) 7 The pregelatinized bio-ink (cells / mL) was mixed with the above-mentioned composite bio-ink at a volume ratio of 1:1. The mixture was transferred into the printing syringe of an extrusion bioprinter (SunP BioMaker 2i professional desktop bio-3D printer, Shangpu Boyuan (Beijing) Biotechnology Co., Ltd.). The printing syringe was placed at 4°C until the mixture turned into a gel. Printing was then performed using a nozzle (300μm diameter). The printer settings were: platform temperature 8°C, printing syringe temperature 15°C, printing pressure 40kPa, and printing speed 5mm / s. The scanning spacing was 1500μm, the single-track width was 500μm, and the layer thickness was 300μm. The resulting pregelatinized mesh was placed in a 2.5% (w / v) CaCl2 aqueous solution and allowed to crosslink at room temperature for 5 minutes. The residual CaCl2 solution was then removed to obtain the printed construct. The results were observed under a microscope (scale bar 500μm). Figure 2 ,Depend on Figure 2 The clear boundaries of the 3D printed construct indicate that the printing ink can maintain a stable shape without collapsing or spreading after extrusion. The organoids within it maintain their shape and grow well, indicating that a stable biological 3D printed liver has been formed.

[0057] Example 3 Functionalized Culture and Detection

[0058] The 3D-printed construct obtained in Example 2 was placed in hepatocyte maturation medium (with the same composition as the hepatocyte expansion medium in Example 1) and cultured for 7 days in an incubator at 37°C and 5% CO2, with the medium changed every two days, to obtain a functionalized bio-3D-printed liver, which was then stored at 4°C for later use. The following detection experiments were then performed.

[0059] 1. ALB (albumin) expression staining experiment

[0060] Take 1 mL of functionalized 3D-printed liver as the sample to be tested, add 5 mL of paraformaldehyde solution (4% PFA) and fix at room temperature for 20 min, wash 3 times with PBS (5 min each time), block with 5% BSA at room temperature with low shaking speed (100 rpm) for 60 min (shaking at low speed during blocking), add 2 mL of diluted ALB primary antibody (abcam, catalog number ab207327, rabbit source, dilution ratio 1:800, diluent is 5% BSA) to ensure that the sample is completely covered, incubate at 4°C overnight, aspirate the primary antibody working solution, wash 3 times with PBS buffer containing 0.02% (v / v) Tween-20 (5 min each time), add 2 mL of the corresponding fluorescently labeled secondary antibody (supplier abcam, catalog number AB150073, fluorescent dye type AlexaFluor) 488 nm, excitation / emission peak ≈ 495 / 519 nm, emitting green fluorescence; dilution ratio 1:800 (diluent: 5% BSA), incubated at room temperature in the dark for 1 h, washed 3 times with PBS (3 min each time), and observed under a fluorescence microscope (scale bar 100 μm). Results are as follows. Figure 3 As shown, the cell distribution area of ​​the functionalized bioprinted liver exhibits green fluorescence (ALB positive signal), which is diffusely distributed in the cytoplasm. The fluorescence signal is uniformly distributed and covers most of the cells of the construct, indicating that most hepatocytes maintain the functional phenotype.

[0061] 2. PAS staining experiment

[0062] Glycogen PAS staining solution (cell-specific) (product model G1360) from Beijing Solarbio Science & Technology Co., Ltd. was used to stain the functionalized 3D-printed liver with glycogen, strictly following the instructions. The purple-red color was used to locate the glycogen substances, and the staining results were observed under a microscope (scale bar 100μm). Figure 4 As shown, rose-red granules and blocky deposits are visible in the cytoplasm of hepatocytes, distributed in most cells, with uniform staining intensity and wide range, indicating that the functionalized bio-3D printed liver has a strong glycogen storage capacity.

[0063] 3. ICG uptake-release experiment

[0064] The uptake and release capacity of functionalized bioprinted livers was tested using indocyanine green (ICG) (catalog number I2633-50MG, brand Sigma). The specific method was as follows: liver maturation medium was discarded, and 1 mg / mL of ICG working solution (diluted with liver maturation medium) was added. The cells were incubated at 37°C and 5% CO2 for 60 min, followed by washing twice with PBS. Immediate microscopic observation revealed that most cells exhibited clearly visible green fluorescence (see...). Figure 5 A, uptake); replace with fresh culture medium (without IGG), continue incubation at 37℃ and 5% CO2 for 6 hours, and fluorescence was significantly reduced under a microscope (see...). Figure 5 B, release), indicating that the functionalized bio-3D printed liver exhibits high ICG uptake and release capacity and high maturity.

[0065] Example 4: Animal Experiment Verification

[0066] Liver-injured mice (strain C57BL / 6Smoc-Fah) from Shanghai Southern Model Biotechnology Co., Ltd. were selected. em1(flox)Smoc The mice required NTBCs to survive in drinking water; liver failure was induced after transplantation and treatment was discontinued. The mice were induced with 3% isoflurane and maintained with 1.5% isoflurane. After anesthesia, the functionalized bio-3D-printed liver from Example 3 was transplanted into the mesenteric region of the aforementioned liver-injured mice. NTBCs were removed one week post-surgery, and the post-operative position in vivo was observed using a stereoscope (scale bar 500 μm) two weeks post-surgery (see [link to original text]). Figure 6 The results showed that the newly formed blood vessels inside the transplant and the host blood vessels formed a continuous network at the interface, indicating vascular integration. This suggests that the transplant can survive and function in the animal.

[0067] Comparative Example 1: Comparison of the maturity of bioprinted livers constructed from different seed cells.

[0068] Cyp2b10 (cytochrome P450 2B10) is involved in the metabolism of drugs and exogenous toxins, and its expression level can reflect the liver's detoxification capacity; Ttr (transthyretin) is synthesized by the liver and is responsible for transporting thyroxine and retinol. Therefore, detecting the transcriptional levels of these two marker genes in mature hepatocytes can be used to compare differences in liver function.

[0069] Using primary mouse hepatocytes as seed cells, 3D bioprinting was performed using the construction method of Example 2 to obtain a 3D-printed liver construct based on hepatocytes. The construct was then functionalized (using the same method as in Example 3) to obtain a 3D-printed liver based on hepatocytes.

[0070] Take 200 μL each of the functionalized bio-3D printed liver from Example 3 (3D printed liver based on liver organoids) and the 3D printed liver based on hepatocytes prepared above (both with a concentration of 1×10⁻⁶). 5All RNA samples (cells / mL) were processed as follows: 1 mL of TRIzol homogenate was added, centrifuged at 12000g for 15 min at 4℃, the supernatant was collected, 0.2 mL of chloroform was added and mixed, and the mixture was allowed to stand for separation. The upper aqueous phase was collected. Isopropanol was added at 1.5 times the volume of the aqueous phase to precipitate RNA, and the RNA was washed three times with 75% ethanol (1 min each time). 20 μL of DEPC was added and the RNA was gently pipetted to ensure complete water dissolution. The purity was determined by Nano Drop assay. The results showed that the concentration of RNA hydrolysate from 3D-printed livers based on liver organoids was 326.52 ng / μL, A260 / A280 = 1.911, and A260 / A230 = 2.298, indicating that the concentration was suitable and the purity was high, making it suitable for direct use in reverse transcription. The concentration of RNA hydrolysate from 3D-printed livers based on hepatocytes was 232.4 ng / μL, A260 / A280 = 2.082, and A260 / A230 = 2.094, indicating that the concentration was suitable and the purity was high, making it suitable for direct use in reverse transcription.

[0071] The obtained RNA hydrolysate was subjected to reverse transcription. The reverse transcription system was: 1 μg RNA hydrolysate, 4 μL 5×RTMasterMix, and Oligo(dT). 18 Add 1 μL of RNase-free water to a final volume of 20 μL; the reverse transcription program is: 25℃ for 5 min → 42℃ for 60 min → 85℃ for 5 min. After the reaction, store the product at -20℃ for later use. Dilute it 10-fold with RNase-free water before use as the cDNA template for qPCR.

[0072] The qPCR reaction system consisted of: 5 μL of 2×SYBR MasterMix, 0.4 μL each of 10 μM forward and reverse primers, and 2 μL of cDNA template. The qPCR program was as follows: 95℃ pre-denaturation for 3 min; 40 cycles of amplification, each cycle consisting of 95℃ denaturation for 10 s and 60℃ annealing and extension for 30 s, with signal acquisition; after the cycles, the temperature was increased in increments of 0.5℃ from 65℃ to 95℃, with each temperature held for 5 s, and signals were acquired and melting curves were plotted. The sequence of the forward primer used for Cyp2b10 qPCR detection was 5'-AAAGTCCCGTGGCAACTTCC-3' (SEQ ID NO.1), and the sequence of the reverse primer was 5'-TTGGCTCAACGACAGCAACT-3' (SEQ ID NO.2). The sequence of the forward primer used for Ttr qPCR detection was 5'-TTGCCTCGCTGGACTGGTA-3' (SEQ ID NO.1). NO.3), the reverse primer sequence is: 5'-TTACAGCCACGTCTACAGCAG-3' (SEQ ID NO.4).

[0073] After preparing the above qPCR reaction system (operate on ice), add it to each well of a 96-well plate, set up 3 technical replicates, place it in a real-time quantitative PCR instrument, run it according to the pre-set program, and perform data analysis. Fibroblasts were used as a negative control. Data analysis is shown below. Figure 7 In terms of the expression of Cyp2b10 and Ttr genes, the 3D-printed liver based on liver organoids was significantly higher than that based on hepatocytes, demonstrating the superiority of the functionalized bio-3D-printed liver of this invention in terms of functional maturity.

[0074] Comparative Example 2: Effects of bio-inks with different compositions on functionalized bio-3D printed livers

[0075] Formulating bio-inks with different compositions:

[0076] Methacrylated gelatin: Add methacrylated gelatin powder to ddH2O preheated to 45–50°C at a ratio of 8% w / v, stir magnetically until completely dissolved, and then filter aseptically through a 0.22 μm filter membrane to obtain the product.

[0077] Gelatin + Sodium alginate: Prepare a 20% w / v gelatin stock solution and a 4% w / v sodium alginate stock solution according to the method in Example 2. Mix them at a volume ratio of 1:1 at 37°C. If microbubbles appear during the process, remove the bubbles by short-term centrifugation (low speed, 4°C) to obtain the final product.

[0078] Gelatin + Sodium Alginate + Hyaluronic Acid: Prepare a 30% w / v gelatin stock solution, a 6% w / v sodium alginate stock solution, and a 6% w / v HA stock solution according to the method in Example 2; mix the above four stock solutions in equal volumes (1:1:1) at 37°C, with the mixing order as follows: first mix gelatin and sodium alginate, and finally add HA. If microbubbles appear during the process, centrifuge briefly (at low speed, 4°C) to remove the bubbles, and mix well to obtain the final product.

[0079] The composite bio-ink in Example 2 was replaced with the above-mentioned bio-ink, while other conditions and parameters remained unchanged. After preparing 3D-printed liver constructs based on liver organoids, they were functionalized and cultured for 7 days (the method is the same as in Example 3) to obtain 3D-printed livers with different bio-inks.

[0080] 200 μL each of the 3D-printed livers with the different inks mentioned above and the functionalized bio-3D-printed liver (composite ink) from Example 3 were used (both with a concentration of 1×10⁻⁶). 5All RNA samples (cells / mL) were processed as follows: 1 mL of TRIzol homogenate was added, centrifuged at 12000g for 15 min at 4℃, the supernatant was collected, 0.2 mL of chloroform was added and mixed, and the mixture was allowed to stand for separation. The upper aqueous phase was collected. Isopropanol was added at 1.5 times the volume of the aqueous phase to precipitate RNA, and the RNA was washed three times with 75% ethanol (1 min each time). 20 μL of DEPC was added and the RNA was gently pipetted to ensure complete water dissolution. The purity was determined by Nano Drop assay.

[0081] The results showed that the concentration of RNA hydrolysate from the functionalized bioprinted liver obtained in methacrylamide gelatin was 166.68 ng / μL, A260 / A280 = 1.894, and A260 / A230 = 2.291, indicating suitable concentration and high purity, suitable for direct use in reverse transcription. The concentration of RNA hydrolysate from the functionalized bioprinted liver obtained in gelatin + sodium alginate was 176.24 ng / μL, A260 / A280 = 2.033, and A260 / A230 = 1.551, indicating suitable concentration and high purity, suitable for direct use in reverse transcription. The concentration of RNA hydrolysate from the functionalized bio-3D printed liver obtained in gelatin + sodium alginate + hyaluronic acid was 289.84 ng / μL, A260 / A280 = 2.058, and A260 / A230 = 1.735, indicating that the concentration was suitable and the purity was high, making it suitable for direct use in reverse transcription. The concentration of RNA hydrolysate from the functionalized bio-3D printed liver obtained in composite bio-ink was 937.68 ng / μL, A260 / A280 = 1.995, and A260 / A230 = 2.165, indicating that the concentration was suitable and the purity was high, making it suitable for direct use in reverse transcription.

[0082] The obtained RNA hydrolysate was subjected to reverse transcription. The reverse transcription system was: 1 μg RNA hydrolysate, 4 μL 5×RTMasterMix, and Oligo(dT). 18 Add 1 μL of RNase-free water to a final volume of 20 μL; the reverse transcription program is: 25℃ for 5 min → 42℃ for 60 min → 85℃ for 5 min. After the reaction, store the product at -20℃ for later use. Before use, dilute 10-fold with RNase-free water to serve as the cDNA template for qPCR.

[0083] The qPCR reaction system consisted of 5 μL of 2×SYBR MasterMix, 0.4 μL each of 10 μM forward and reverse primers, and 2 μL of cDNA template. The qPCR reaction program was as follows: 95℃ pre-denaturation for 3 min; 40 cycles of amplification, with each cycle consisting of 95℃ denaturation for 10 s and 60℃ annealing and extension for 30 s, followed by signal acquisition; after the cycles, the temperature was increased in increments of 0.5℃ from 65℃ to 95℃, with each temperature held for 5 s, and signals were acquired and melting curves were plotted. The sequence of the forward primer used for Alb qPCR detection was 5'-CGCCAACTGTGACAAATCCC-3' (SEQ ID NO. 5), and the sequence of the reverse primer was 5'-GCCTTTCAAATGGTGGCAGG-3' (SEQ ID NO. 6). The forward and reverse primers used for Ttr qPCR detection were the same as those used for Ttr qPCR detection in Comparative Example 1.

[0084] After preparing the above qPCR reaction system (operate on ice), add it to each well of a 96-well plate, set up 3 technical replicates, place it in a real-time quantitative PCR instrument, run it according to the pre-set program, and perform data analysis. See the data analysis below. Figure 8 In the composite bio-ink selected in this invention, the expression level of Alb is significantly higher than that of the single methacrylamide gelatin system, the sodium alginate gelatin composite system, and the sodium alginate gelatin hyaluronic acid composite system. The expression trend of Ttr is consistent with that of Alb, reaching the highest level in the bio-ink group of this invention. This indicates that hepatocytes in the printed body can maintain secretory and synthetic functions in the selected composite ink system, and their functional maturity is significantly better than that of traditional inks. Therefore, the formulation of this invention has a significant advantage in maintaining the long-term activity and functional maturity of hepatocytes, surpassing existing single-component or two-component inks.

[0085] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0086] 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 a functionalized bio-3D printed liver based on organoids, characterized in that, Includes the following steps: (1) Primary hepatocytes were induced in vitro to obtain liver organoid suspension; (2) Preparation of composite bio-ink; (3) The liver organoid suspension was mixed with the composite bio-ink and the mixture was transferred to a 3D bioprinter for printing to obtain a pre-gelled mesh; (4) Cross-link the pregel mesh at room temperature and then perform functional maturation culture to obtain a functionalized bio-3D printed liver.

2. The method for constructing a functionalized bio-3D printed liver based on organoids according to claim 1, characterized in that, The liver organoid suspension described in step (1) was prepared by the following method: primary hepatocytes were digested into single cells, and then subjected to a reaction at a ratio of 5 × 10⁻⁶ cells / mL. 6 The cells were seeded into the culture medium at a density of cells / mL and placed in hydrophobic culture dishes for 2 days at 37°C, 5% carbon dioxide, 90% humidity, and 100 rpm.

3. The method for constructing a functionalized bio-3D printed liver based on organoids according to claim 1, characterized in that, The composite bio-ink described in step (2) consists of: 10% (w / v) gelatin, 2% (w / v) sodium alginate, 8% (w / v) methacrylamide gelatin and 2% (w / v) hyaluronic acid.

4. The method for constructing a functionalized bio-3D printed liver based on organoids according to claim 1, characterized in that, Step (3) involves transferring the mixture to the 3D bioprinter for printing, specifically including the following operations: transferring the mixture to the 3D bioprinter, placing the printing syringe at 4°C, and printing with the nozzle when the mixture turns into a gel to obtain a pre-gelled mesh.

5. The method for constructing a functionalized bio-3D printed liver based on organoids according to claim 4, characterized in that, The parameters of the 3D bioprinter are set as follows: platform temperature 8℃, printing cylinder temperature 15℃, printing pressure 40kPa, and printing speed 5mm / s. The printing process has a scanning spacing of 1500μm, a single-track width of 500μm, and a layer thickness of 300μm.

6. The method for constructing a functionalized bio-3D printed liver based on organoids according to claim 1, characterized in that, Step (4) includes the following steps: (a) Crosslink the pregel mesh with 2.5% (w / v) CaCl2 solution at room temperature for 5 min, remove the residual CaCl2 solution, and obtain the 3D printed construct; (b) The printed construct was placed in hepatocyte maturation medium and cultured at 37°C and 5% CO2 for 7 days, with the medium being changed every two days during the period.

7. A functionalized bio-3D printed liver, characterized in that, Obtained by the construction method according to any one of claims 1-8.

8. Any of the following applications of the functionalized bio-3D printed liver obtained by the construction method according to any one of claims 1-6 or the functionalized bio-3D printed liver according to claim 7: 1) Preclinical drug testing; 2) Materials used in regenerative medicine or in vivo transplantation; 3) Used for research on bioartificial livers or liver function compensation; 4) Pathological research on liver diseases; 5) New drug research and development.

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