Injectable decellularized matrix-liver organ three-dimensional compound as well as preparation method and application thereof

Abstract

The invention discloses an injectable decellularized matrix-liver organ three-dimensional compound as well as a preparation method and application thereof. The three-dimensional compound comprises decellularized matrix fibers and liver organs which form a pasty compound, the length of the decellularized matrix fiber is 0.2-2mm, and the decellularized matrix fiber is obtained by selecting mammalian dermis, performing epidermis removal and degreasing, performing soaking treatment with alkali and a nonionic surfactant in sequence, then performing decellularization, and performing freeze-drying and ball milling. The liver organ is obtained by placing bile in matrigel for culture, amplification and induction. According to the invention, a Mini-liver complex with injectability, self-adhesion, biological activity and metabolic function is formed, unified integration of a material scaffold, signal delivery and cell function is realized, and an innovative solution with remarkable clinical potential is provided for local repair after hepatic resection and hepatic failure.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical engineering and regenerative medicine, and relates to an injectable decellularized matrix-liver organoid three-dimensional composite and a preparation method and application thereof, in particular to an injectable "Mini-liver" biomaterial based on the combination of decellularized extracellular matrix short fibers (DEFs) and bile-derived liver organoids (BDLOs), and the application thereof in the recovery of liver function after liver resection and the treatment of acute liver failure. BACKGROUND

[0002] Liver failure and liver dysfunction after liver resection are common critical conditions in clinical practice. The existing treatment methods mainly include liver transplantation, artificial liver support and stem cell transplantation. However, these techniques have obvious shortcomings: liver transplantation is limited by donor shortage and immune rejection, and long-term postoperative immunosuppression increases the risk of infection and tumor recurrence; artificial liver mainly removes toxins through blood perfusion, and cannot restore the synthetic metabolic function of the liver; cell transplantation and organoid transplantation can provide partial metabolic support, but have problems such as high early cell mortality (>70%), low graft retention rate (<5%) and unstable function.

[0003] To improve the survival rate and functional stability of transplanted cells, researchers have begun to try to use biomaterials to construct a three-dimensional microenvironment that can support the growth of liver cells. For example, the current scientific community attempts to use collagen, alginate, gelatin methacrylate (GelMA) and other hydrogels as organoid or hepatocyte carriers. Although these materials have good biocompatibility, they generally have the following defects: (1) mismatched mechanical properties: the low viscoelasticity of hydrogels cannot withstand the dynamic mechanical environment on the liver surface, and they are easily washed off after implantation; (2) limited diffusion of nutrients and oxygen: the interior of the gel is dense, leading to central cell necrosis; (3) short release time of growth factors: the release duration is usually only 12-24h, making it difficult to maintain early regenerative signals; (4) complex clinical operation: in vitro solidification or photocrosslinking is required, which is not suitable for minimally invasive application.

[0004] Decellularized extracellular matrix (ECM) materials that have appeared in recent years have shown excellent biological activity in tissue repair, and are therefore considered to be ideal natural scaffold materials. However, their common forms are usually in the form of sheets, blocks or gels, which have poor processability and flowability, lack injectability and tissue adhesion, and are difficult to implant through minimally invasive methods. When they are applied to the preparation of liver organoids, they often cannot adapt to the irregular shape of the liver surface.

[0005] Therefore, there is an urgent need to develop a new composite system that has injectability, tissue adhesion, biological activity and bidirectional regeneration regulation ability, in order to achieve rapid bridging and long-term regeneration of liver function. SUMMARY

[0006] The first objective of this invention is to overcome the shortcomings of the prior art and provide a method for preparing an injectable decellularized matrix-liver organoid three-dimensional complex.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing a decellularized matrix-liver organoid three-dimensional complex includes the following steps: Mammalian dermis was selected, and after desquamation and defatting, it was successively soaked in alkali and nonionic surfactants, then decellularized, and obtained decellularized matrix fibers after freeze-drying and ball milling. Bile was cultured in a matrix gel, then amplified using human liver amplification medium and induced using differentiation medium to form liver organoids; The decellularized matrix fibers and liver organoids were mixed and incubated at 37°C for 5-30 min to form the decellularized matrix-liver organoid three-dimensional complex.

[0008] Preferably, the length of the decellularized matrix fibers is 0.2-2 mm.

[0009] Preferably, during the soaking treatment, the alkali used is sodium carbonate with a mass fraction of 1%-3%; and the nonionic surfactant used is Triton X-100 with a mass fraction of 0.1%-0.5%.

[0010] Preferably, the soaking temperature is 30-40℃ and the time is 2-4 hours.

[0011] As a preferred method, decellularization is performed by shaking a 0.1-1% deoxycholic acid solution at 25-37°C for 12-20 hours.

[0012] Preferably, Matrigel is used as the matrix gel, h-EM is used as the human liver amplification medium, and the amplification time is 5-7 days; h-DM is used as the differentiation medium, and the induction time is 3-5 days.

[0013] Preferably, the ratio of decellularized matrix fibers to liver organoids is 10-100 mg: (1.5 × 10⁻⁶ mg / mL). 6 -1.5×10 7 cells).

[0014] A second objective of this invention is to provide a decellularized matrix-liver organoid three-dimensional complex, prepared using the method described above. In this three-dimensional complex, the decellularized matrix fibers are directly injected onto the liver surface via minimally invasive injection, immobilizing the liver organoid and forming a microenvironment. Through a dual mechanism of exogenous organoid metabolic function and matrix regulation of the host regenerative environment, liver function recovery, vascular remodeling, and fibrosis inhibition are achieved.

[0015] A third objective of this invention is to provide the application of the above-described decellularized matrix-liver organoid three-dimensional complex in the preparation of organ repair materials.

[0016] Preferably, during organ repair, the administration method is local injection or spraying.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a composite system with a technological concept different from existing liver tissue repair strategies. By synergistically constructing decellularized matrix short fibers (DEFs) and bile-derived liver organoids (BDLOs) into an injectable paste-like complex, it achieves integrated structural support, bioactivity, and metabolic function. This composite not only overcomes the limitations of traditional dECM materials in terms of morphology and application, but also demonstrates a significant synergistic effect in maintaining cell function and tissue regeneration mechanisms. Specifically, processing dECM into a short fiber morphology significantly improves fluidity and specific surface area while maintaining the original tissue's bioactivity, allowing for smooth injection through conventional needles and self-adhesion on the liver surface to form a stable three-dimensional scaffold layer. Its microfiber network provides mechanical support while continuously releasing various regenerative factors such as VEGF, HGF, FGF, and IGF, reconstructing local vascularization and signal microenvironment. When DEFs and BDLOs are co-assembled, the release of regenerative factors in the material microenvironment and the metabolic activity of organoids form a mutually reinforcing feedback, promoting angiogenesis, inhibiting inflammation and fibrosis, and activating the host PPARα-YAP signaling pathway, thereby promoting hepatocyte regeneration from the energy metabolism level and effectively overcoming the shortcomings of existing technologies where BDLOs are limited by the in vivo microenvironment. Attached Figure Description

[0018] Figure 1 The preparation process and structural characterization of decellularized dermal short fibers (DEFs). (A) Schematic diagram of the preparation process of decellularized dermal short fibers; (B) Scanning electron micrograph of DEFs, showing fiber morphology and porous structure characteristics.

[0019] Figure 2 Figure 1 shows the results of DEFs promoting structural remodeling and functional maturation of bile-derived liver organoids (BDLOs). (A) Bright-field micrographs of BDLOs in the DEF-treated group and the control group at 48 h and 96 h of induction culture; (B, C) Changes in organoid wall thickness at 96 h and their quantitative analysis; (D, E) Immunofluorescence staining and quantitative results of HNF4A and ALB; (F, G) Immunofluorescence staining and quantitative results of Ki67 and CYP3A4; Scale bar 100 μm.

[0020] Figure 3Schematic diagram of the construction and injection adhesion of the Mini-liver complex. (A) Schematic diagram of the process of forming a three-dimensional network Mini-liver by combining DEFs and BDLOs; (B) Intraoperative image of Mini-liver delivered to the liver surface by injection; (C) Scanning electron micrograph of the distribution and adhesion of Mini-liver on the material surface, where the left is the original image with a scale bar of 50 μm and the right is the magnified image with a scale bar of 10 μm.

[0021] Figure 4 The results of in vitro angiogenesis and anti-fibrosis assays are shown. (A) Images of HUVEC tube formation experiments, comparing tube formation in the control group and the DEF-conditioned medium treatment group; (B) Statistical bar chart of HUVEC tube formation experiments; (C) Fluorescent staining and quantitative analysis of α-SMA and COL1 in LX2 cells; (D) qPCR results of α-SMA, COL1, TGF-β, SMAD2, SMAD3, COL3 and CCN2 in LX2 cells.

[0022] Figure 5 The therapeutic effect of Mini-liver in a mouse model of 70% hepatectomy. (A) Schematic diagram of animal grouping and drug administration process; (B) Representative image of intraoperative injection on the liver surface in the Mini-liver group; (C) Curves of ALT, AST, and TBIL over time in each group; (D) Survival curves in each group; (E) Images of liver tissue stained with HE and CD31, scale bar 100 μm; (F) Quantitative analysis of CD31 staining results in liver tissue.

[0023] Figure 6 Mini-liver promotes hepatocyte regeneration by activating the PPARα and YAP pathways. (A) GSEA analysis showed that the PPARα signaling pathway was significantly upregulated in the treatment group; (B, C) Immunohistochemical staining and quantitative analysis of PPARα and YAP in liver tissue; (D) Schematic diagram of the mechanism: Mini-liver simultaneously activates the PPARα / YAP / TAZ axis, promotes the expression of regeneration-related genes, and accelerates liver tissue repair. Detailed Implementation

[0024] The present invention will be further described below with reference to the accompanying drawings and embodiments. The raw materials used in the embodiments can be commercially available or prepared by conventional methods.

[0025] Example 1: Preparation and characterization of decellularized dermal short fibers (DEFs) like Figure 1As shown in Figure A, healthy adult pig skin (source: Experimental Animal Center, Zhejiang University) was selected. After removing the epidermis and subcutaneous fat, it was cut into small pieces of 1cm × 1cm × 0.5cm. The samples were sterilized by shaking in a 1% sodium hypophosphite and 5% peracetic acid solution at room temperature for 3 hours. Subsequently, they were sequentially placed in a 2% Na₂CO₃ + 0.2% Triton X-100 solution (37℃ × 3 hours) and a 0.1% HEPES + 0.2% EDTA + 1% deoxycholic acid solution (150 rpm × 16 hours) for decellularization. After washing to neutral, the samples were freeze-dried, ball-milled (8000 rpm × 30 minutes), and short fibers with a length of 0.2-2 mm were screened. The fibers were sterilized by 17.6 kGy gamma ray and then stored. The length and pore structure were observed using scanning electron microscopy. Figure 1 As shown in B in the figure, SEM revealed that DEFs have a regular loose structure, which facilitates cell loading.

[0026] Example 2: Establishment of Bile-Derived Liver Organoids (BDLOs) Collect 5 mL of bile sample from patients undergoing ERCP and transport on ice for up to 1 hour before processing. Centrifuge the sample (400×g×5min), discard the supernatant, wash the precipitate with PBS + 1% BSA, and filter (70 μm). Take 5×10 4 Cells were suspended in Matrigel, and 30 μL was added to the center of a 24-well plate and gelled at 37°C for 10 min. Human liver amplification medium (h-EM) was added, and the cells were cultured at 5% CO2 and 37°C for 7 days, with medium changes every 48 hours. Then, differentiation medium (h-DM) was used for induction for 3-5 days. Immunostaining showed that the organoids expressed SOX9, EpCAM, HNF4A, and CYP3A4, forming spherical clusters with a diameter of 60-120 μm. The culture medium formulations used in this invention are shown in Table 1. Table 1. Formulations of human liver amplification and differentiation media The prepared DEFs were co-cultured with the formed BDLOs to observe the promoting effect of DEFs on BDLOs. The experimental group used BDLOs + DEFs, while the control group used only BDLOs. Both groups were cultured in the same culture medium. Observations and photographs were taken under a microscope at different time points during co-culture. Furthermore, immunofluorescence staining of the organoids was performed using specific antibodies such as liver-specific antibodies ALB, HNF4A, and CYP3A4. Image analysis software was used to quantitatively analyze the fluorescence intensity or positive cell area of ​​the fluorescence images. The results are as follows: Figure 2 As shown, after DEF treatment, the expression of liver-specific markers in organoids was enhanced to varying degrees, and they exhibited a denser three-dimensional structure. The outer structure of the organoids was thicker and more uniform, suggesting that they can promote organoid structural remodeling and promote the differentiation of organoids into mature hepatocytes.

[0027] Example 3: Construction and Injection Performance Testing of Mini-liver Complex Weigh 20 mg of sterile DEFs into a 1.5 mL tube, add 200 μL of solution containing 1.5 × 10⁻⁶ ppm. 7 A BDLO suspension of cells / mL was gently mixed by pipetting and incubated at 37°C for 5 min to form a primary adhesion complex, which is the Mini-liver complex. Figure 3 As shown, the complex was injected using a 22G needle (0.7 mm inner diameter) at a flow rate of 0.1-0.2 mL / s, and the continuity and needle resistance were observed. The results showed that the complex could be injected smoothly and remained uniform, and the Mini-liver could adhere smoothly to the liver surface.

[0028] Example 4: Evaluation of the effects of DEFs on endothelial cell function and LX2 cell activation status This embodiment evaluated the biological responses of endothelial cells and hepatic stellate cells after DEF treatment. Figure 4 As shown, human umbilical vein endothelial cells (HUVECs) were co-cultured with DEFs, and microscopic images of capillary formation were acquired at 2h, 4h, and 6h of culture (scale bar: 100μm). Subsequently, relevant parameters of capillary formation (including tubular structure length) were quantitatively analyzed (n=3 per group). Simultaneously, LX2 cells were divided into a control group, a TGF-β stimulation group, and a TGF-β+DEFs treatment group, and immunofluorescence staining for Collagen I and α-SMA was performed on each group (scale bar: 100μm). The average fluorescence intensity was then quantitatively compared (n=4 per group). Furthermore, qRT-PCR was used to measure the mRNA levels of α-SMA, COL1, TGF-β, SMAD2, SMAD3, COL3, and CCN2 in the three groups of LX2 cells (n=4 per group). The experimental results showed that under the culture conditions, the capillary formation-related indicators of endothelial cells in the DEFs-treated group showed an upward trend, and the expression of fibrosis-related proteins and genes in LX2 cells showed a downward trend, providing data support for the role of DEFs.

[0029] Example 5: Validation of a 70% Hepatectomy Model in Mice This embodiment validates the effect of Mini-liver implantation on an acute large-scale hepatectomy model. In this experiment, FRG mice were used as experimental animals and divided into a control group, a model group, a DEFs group, and a Mini-liver (organoid + DEFs) group according to the treatment method. FRG mice underwent 70% hepatectomy. In the Mini-liver group, 100 μL of the composite was uniformly injected onto the liver surface during the operation, while the DEFs group only received DEF suspension. In vivo follow-up was conducted for 14 consecutive days to monitor the status and function of the implanted material in the mice.

[0030] Experimental results are as follows Figure 5 In the model group mice, significantly elevated ALT, AST, and TBIL levels were observed after hepatectomy, while the DEFs and Mini-liver groups showed varying degrees of decline. The Mini-liver group exhibited even lower values ​​for these indicators (n=6 per group). Survival rate records showed the lowest survival rate in the model group, while both the DEFs and Mini-liver groups showed higher survival rates, with the Mini-liver group's survival rate curve falling within the optimal range. Quantitative analysis of HE and CD31 staining results revealed relatively improved tissue structure in both the DEFs and Mini-liver groups, with the Mini-liver group showing higher levels of vascular-related indicators (n=6 per group).

[0031] Example 6 Mechanism Verification and Signal Path Analysis RNA-seq analysis was performed on mouse liver tissue from Example 5, such as... Figure 6 As shown, KEGG enrichment results revealed upregulation of the PPARα pathway and Hippo-YAP signaling. Immunohistochemistry showed that compared to the model group, treatment with Mini-liver (organoid + DEFs) increased YAP nuclear localization and PPARα expression, suggesting that this complex promotes regeneration through PPARα / YAP.

[0032] The materials used in this invention are widely available, low in cost, and can be prepared in batches and stored for a long time (stable for 6 months at -20°C). After injection, this complex self-adheres to the liver surface without the need for sutures, making it suitable for post-hepatectomy treatment and the treatment of acute liver failure, demonstrating significant potential for clinical and industrial translation.

[0033] In summary, the decellularized matrix-liver organoid composite system provided by this invention combines injectability, bioadhesion, and a biaxial regeneration mechanism, which can significantly promote liver function recovery and improve survival rate, providing a new translational approach for regenerative treatment of liver diseases.

[0034] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a decellularized matrix-liver organoid three-dimensional complex, characterized in that, Includes the following steps: Mammalian dermis was selected, and after desquamation and defatting, it was successively soaked in alkali and nonionic surfactants, then decellularized, and obtained decellularized matrix fibers after freeze-drying and ball milling. Bile was cultured in a matrix gel, then amplified using human liver amplification medium and induced using differentiation medium to form liver organoids; The decellularized matrix fibers and liver organoids were mixed and incubated at 37°C for 5-30 min to form the decellularized matrix-liver organoid three-dimensional complex.

2. The preparation method according to claim 1, characterized in that, The length of the decellularized matrix fibers is 0.2-2 mm.

3. The preparation method according to claim 1, characterized in that, During the soaking treatment, sodium carbonate is used as the alkali, with a mass fraction of 1%-3%; the nonionic surfactant used is Triton X-100, with a mass fraction of 0.1%-0.5%.

4. The preparation method according to claim 3, characterized in that, The soaking temperature is 30-40℃ and the time is 2-4 hours.

5. The preparation method according to claim 1, characterized in that, Decellularization was performed by shaking a 0.1-1% deoxycholic acid solution at 25-37°C for 12-20 hours.

6. The preparation method according to claim 1, characterized in that, Matrigel was used as the matrix gel, h-EM was used as the human liver amplification medium, and the amplification time was 5-7 days; h-DM was used as the differentiation medium, and the induction time was 3-5 days.

7. The preparation method according to claim 1, characterized in that, The ratio of decellularized matrix fibers to liver organoids was 10-100 mg: (1.5 × 10⁻⁶) 6 -1.5×10 7 cells).

8. A decellularized matrix-liver organoid three-dimensional complex, characterized in that, It is prepared by the method described in any one of claims 1-7.

9. The application of the decellularized matrix-liver organoid three-dimensional complex as described in claim 8 in the preparation of organ repair materials.

10. The application according to claim 9, characterized in that, During organ repair, the administration method is local injection or spraying.

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