Biological mixed decellularized liver cancer para-carcinoma tissue hydrogel as well as preparation method and application thereof

Abstract

The invention is suitable for the technical field of biomedical materials, and provides a biological mixed decellularized liver cancer para-carcinoma tissue hydrogel as well as a preparation method and application thereof. The prepared decellularized hydrogel is wide in raw material source, clinically available waste such as human liver cancer para-carcinoma tissue blocks is converted into a scaffold material, the problem that scaffold sources are scarce is effectively solved, and resource utilization of clinical waste is achieved; meanwhile, the decellularized hydrogel completely retains a natural extracellular matrix, is low in immunogenicity and excellent in biocompatibility, can provide a highly bionic structural platform for cells (such as hepatocytes derived from human induced pluripotent stem cells), and fully meets the requirements of cell adhesion, growth and proliferation; besides, exogenous regeneration active substances such as mesenchymal stem cell source exosomes can be effectively integrated, the capabilities of regulating inflammation, promoting tissue repair and improving cell survival are remarkably enhanced, and a novel and effective treatment mode is provided for acute hepatic failure treatment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical materials, and particularly relates to a biological mixed decellularized liver cancer paracancer tissue hydrogel as well as a preparation method and application thereof. BACKGROUND

[0002] Acute liver failure (ALF) is a severe clinical disease with rapid disease progression and high mortality rate. The main features are rapid deterioration of liver function and massive necrosis of hepatocytes. At present, liver transplantation is the main means to deal with this disease. However, the severe shortage of donor organs and various risks during the transplantation process greatly limit the wide application of liver transplantation technology. Under this background, tissue engineering technology has gradually become a promising alternative strategy for the treatment of ALF. Among them, the decellularized scaffold removes the cellular components and retains the natural extracellular matrix (ECM), which provides an ideal platform for liver tissue regeneration. This kind of scaffold not only can support cell adhesion and proliferation, but also is rich in bioactive molecules that guide cell behavior and tissue structure.

[0003] Clinically obtained tissue samples are often regarded as medical waste, but in fact these tissue samples can be a potential source of natural scaffolds. In particular, the tumor surrounding tissue treated by mild decellularization can retain a large number of bioactive components while maintaining low immunogenicity, and has good clinical application prospects.

[0004] Mesenchymal stem cells (MSCs) are a kind of adult stem cells with self-renewal and multi-directional differentiation potential, which play an important role in tissue repair. Integrating MSC-derived exosomes into hydrogel systems can effectively enrich endogenous regenerative cytokines. These exosomes carry a variety of bioactive substances, which can mediate tissue repair, regulate inflammatory response, and promote cell survival through a paracrine mechanism.

[0005] Although tissue engineering shows potential in the treatment of ALF, the current treatment strategies still have many obvious limitations. On the one hand, the autologous source of decellularized scaffolds is scarce, which limits its wide application; on the other hand, the scaffold simply seeded with hepatocytes lacks an efficient regeneration promotion mechanism and does not have sufficient anti-inflammatory capacity, thereby affecting the treatment effect. Therefore, developing new treatment strategies to improve the survival rate of ALF patients and restore liver function has become an important problem to be solved.

[0006] Therefore, the present application provides a biological mixed decellularized liver cancer paracancer tissue hydrogel as well as a preparation method and application thereof. SUMMARY

[0007] The present application aims to provide a biological mixed decellularized liver cancer paracancerous tissue hydrogel, a preparation method and application thereof, and aims to solve the problems raised in the above background.

[0008] The purpose of the present application is achieved by the following technical solutions: A biological mixed decellularized liver cancer paracancerous tissue hydrogel, comprising a decellularized hydrogel and mesenchymal stem cell-derived exosomes, 1 mg of mesenchymal stem cell-derived exosomes per 1 cm 3 The decellularized hydrogel is loaded with 1 mg of mesenchymal stem cell-derived exosomes; the decellularized hydrogel is formed after the human liver cancer paracancerous tissue is treated by decellularization and enzyme digestion; the mesenchymal stem cell-derived exosomes are obtained from the mesenchymal stem cell culture supernatant by differential centrifugation.

[0009] Further, the preparation steps of the decellularized hydrogel are as follows: Take a piece of human liver cancer paracancerous tissue, immerse it in a solution containing 1% Triton-X 100, and incubate it at 4°C; rinse the tissue repeatedly with sterile PBS to complete the decellularization process and obtain a decellularized extracellular matrix; place the decellularized extracellular matrix in a centrifuge tube containing a digestion solution and incubate it in a 37°C water bath for 2 hours; then filter the digested solution through a cell filter and incubate the filtered digestion solution at 37°C for 1 hour to form a decellularized hydrogel; The digestion solution consists of 0.25% trypsin and 0.05% collagenase type II.

[0010] Further, the extraction steps of the mesenchymal stem cell-derived exosomes are as follows: Collect rat bone marrow fluid, centrifuge at 300-400g for 5 minutes, and discard the supernatant; resuspend the bone marrow cell pellet in serum-free DMEM basal medium, then supplement 10% fetal bovine serum and 1% penicillin-streptomycin to the medium to prepare mesenchymal stem cell complete medium; inoculate the cells into a sterile culture dish and incubate at 37°C, 5% CO2; when the cells reach 90% confluence, first collect the mesenchymal stem cell complete medium, then add 0.25% trypsin to the culture dish to digest the cells, separate them from the culture dish and pass them on; centrifuge the collected mesenchymal stem cell culture medium at 400g for 10 minutes, transfer the supernatant to a sterile centrifuge tube, and centrifuge at 2000g for 10-20 minutes; centrifuge the supernatant at 10000-20000g for 30 minutes, collect the exosome-containing pellet and resuspend it in PBS.

[0011] A three-dimensional hepatocyte culture system comprising the biological mixed decellularized liver cancer paracancerous tissue hydrogel and human induced pluripotent stem cell-derived hepatocytes as described above.

[0012] Further, the liver cell three-dimensional culture system is used for promoting liver cell proliferation or maintaining liver cell function in vitro.

[0013] Further, the liver cell three-dimensional culture system is used for establishing an in vitro liver injury model.

[0014] Further, the construction steps of the liver cell three-dimensional culture system are as follows: The human induced pluripotent stem cell-derived liver cells are co-cultured with the culture medium supplemented with the biological mixed decellularized liver cancer para-cancer tissue hydrogel, and the culture condition is 37 DEG C, 5% CO2, and the liver cell three-dimensional culture system is obtained. The culture medium is DMEM culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin.

[0015] Further, in the liver cell three-dimensional culture system, 1 cm 3 The decellularized hydrogel carries 1x10 7 The human induced pluripotent stem cell-derived liver cells.

[0016] The biological mixed decellularized liver cancer para-cancer tissue hydrogel is used for preparing a preparation for culturing liver cells in vitro or establishing an in vitro liver injury model.

[0017] Compared with the prior art, the beneficial effects of the present application are: 1. The decellularized hydrogel prepared by the present application has a wide source of raw materials, and the clinical waste (such as human liver cancer para-cancer tissue block) is converted into a scaffold material, effectively solving the problem of scarcity of scaffold sources, and realizing the resource utilization of clinical waste.

[0018] 2. The decellularized hydrogel prepared by the present application retains the natural extracellular matrix, has low immunogenicity, and has excellent biocompatibility. The hydrogel can provide a highly biomimetic structural platform for cells (such as human induced pluripotent stem cell-derived liver cells), and fully meet the needs of cell adhesion, growth and proliferation.

[0019] 3. The decellularized hydrogel prepared by the present application can effectively integrate exogenous living active substances (such as mesenchymal stem cell-derived exosomes), and through the synergistic effect of the two, the ability to regulate inflammation, promote tissue repair and improve cell survival is significantly enhanced, providing a new and effective treatment for acute liver failure. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 It is a schematic diagram of the construction of the iPSC-Heps three-dimensional co-culture system based on the exosome-loaded decellularized hydrogel.

[0021] Figure 2For characterization of the decellularized hydrogel; wherein: a is degradation experiment; b is erosion experiment; c is swelling experiment; d is viscoelasticity experiment.

[0022] Figure 3 For biocompatibility of the decellularized hydrogel and the exosome-loaded decellularized hydrogel; wherein: a is live-dead staining; b is CCK8 experiment.

[0023] Figure 4 For therapeutic effect of the exosome-loaded decellularized hydrogel; wherein: a is H&E staining; b is TUNEL staining; c is survival rate; d is liver function index (ALT, AST).

[0024] Figure 5 For liver repair effect of the exosome-loaded decellularized hydrogel; wherein: a is immunofluorescence (TNF-a); b is immunofluorescence (IL-6); c is TNF-a, IL-6, COX2 gene expression level. DETAILED DESCRIPTION

[0025] In order to have a clearer understanding of the technical features, objectives and beneficial effects of the present application, the technical solutions of the present application will be described in detail below, but it should not be understood as limiting the scope of the present application. Unless otherwise specified, the methods used in the present application are conventional methods in the technical field. In the present application, the materials, reagents or instruments used are not specified by the manufacturer, but are conventional products that can be obtained by market purchase.

[0026] The specific implementation of the present application will be described in detail below in combination with specific examples.

[0027] Example 1: Construction of iPSC-Heps three-dimensional co-culture system based on exosome-loaded dECM hydrogel Figure 1 ); Step 1: Preparation of decellularized (dECM) hydrogel (1) Take human liver cancer para-carcinoma tissue block, immerse it in a solution containing 1% Triton-X 100, and incubate at 4°C to destroy the cell membranes of cells in the tissue and release cell contents; (2) Wash the above-mentioned tissue repeatedly (at least three times) with a large amount of sterile PBS to eliminate residual detergents, cell debris and dissolved substances, complete the decellularization treatment, and obtain a decellularized extracellular matrix.

[0028] (3) Put the decellularized extracellular matrix into a centrifuge tube containing a digestive solution (composed of 0.25% trypsin and 0.05% collagenase type II), incubate in a 37°C water bath for 2h; then filter the digested solution through a 100μm cell filter to remove undigested tissue blocks and large particulate matter.

[0029] (4) The filtered digestive juice was incubated at 37 °C for 1 h to form a decellularized (dECM) hydrogel.

[0030] Step 2: Extraction of mesenchymal stem cell-derived exosomes; (1) The bone marrow fluid of rats (purchased from Spibio (Suzhou) Biotechnology Co., Ltd.) was collected and centrifuged at low speed (about 300 g for 5 min) to remove the supernatant to eliminate blood components. The bone marrow cell pellet was resuspended in serum-free DMEM basal medium, and then 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P-S) were added to the medium to prepare mesenchymal stem cell complete medium. The above cells were inoculated into a sterile culture dish and cultured at 37 °C, 5% CO2. Once the cells reached 90% confluence, the mesenchymal stem cell complete medium was collected, and 0.25% trypsin was added to the culture dish to digest the cells, which were then separated from the culture dish and passaged.

[0031] (2) The collected mesenchymal stem cell medium was centrifuged at 400 g for 10 min to remove suspended cells and cell debris; the supernatant was transferred to a sterile centrifuge tube and centrifuged at 2000 g for 20 min to remove larger particulate contaminants.

[0032] (3) The supernatant was ultracentrifuged at 20,000 g for 30 min, and the precipitate containing exosome particles was collected and resuspended in PBS.

[0033] Step 3: Construction of iPSC-Heps three-dimensional co-culture system based on exosome-loaded dECM hydrogel; Human induced pluripotent stem cell-derived hepatocytes (iPSC-Heps) were co-cultured with dECM hydrogel prepared in step 1 and exosomes obtained in step 2 in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P-S) (37 °C, 5% CO2) to construct a three-dimensional (3D) co-culture system. In this system, 1 cm 3 dECM hydrogel loaded with 1 x 10 7 iPSC-Heps cells and 1 mg of exosomes.

[0034] Example 2: In vitro stability characterization of dECM hydrogel; To explore the effect of enzymes on hydrogel degradation, the dECM hydrogel was immersed in PBS containing 5 U / mL and 10 U / mL collagenase type II, respectively, and the degradation experiment was carried out at 37 °C. As the incubation time in the collagenase type II solution increased, the dECM hydrogel gradually degraded, and the degradation rate of the 10 U / mL collagenase group was faster than that of the 5 U / mL group, indicating that the higher the collagenase concentration, the faster the hydrogel degradation (Fig. 2). Figure 2(a) To investigate the effect of pH on the erosion of dECM hydrogel, the erosion of dECM hydrogel was observed under pH=5.5 and pH=7.4 conditions. Figure 2 Figure b shows that the erosion rate of the dECM hydrogel was slow under both pH conditions, indicating that the dECM hydrogel maintains stability in the absence of the enzyme. Swelling assays showed that the dECM hydrogel exhibited rapid swelling behavior within 6 hours, with a swelling rate approaching 1200%. Figure 2 (c). Furthermore, viscoelasticity analysis of the dECM hydrogel showed that G' and G'' values ​​remained relatively stable within a strain range of 1% to 10%, indicating the existence of a linear viscoelastic region in the dECM hydrogel. Figure 2 (d).

[0035] Example 3: Biocompatibility of dECM hydrogels and dECM hydrogels loaded with exosomes; To evaluate the biocompatibility of dECM hydrogels and the effect of exosome loading on cell growth, iPSC-Heps were cultured in two-dimensional (2D) and three-dimensional (3D) environments, and cell viability was assessed. 2D culture (dECM hydrogel group): iPSC-Heps were seeded onto the surface of dECM hydrogels and cultured. 3D culture (dECM hydrogel group loaded with exosomes): Based on the 3D co-culture system constructed in Example 1. Live / dead cell staining results showed ( Figure 3 (a) When iPSC-Heps were cultured on the surface of dECM hydrogel for up to 3 days, green fluorescence (in live cells) was continuously and densely distributed, and the cells maintained good viability; CCK8 assay results showed ( Figure 3 (b) Cell viability in the 3D culture group was higher than that in the 2D culture group. These results indicate that the dECM hydrogel has low toxicity to iPSC-Heps and excellent biocompatibility; moreover, compared with 2D culture, the exosome-loaded dECM hydrogel can further promote cell proliferation in 3D culture, demonstrating the potential of exosome-loaded dECM hydrogel in promoting cell growth.

[0036] Example 4: The therapeutic effect of exosome-loaded dECM hydrogel on acute liver failure; To investigate the therapeutic effect of exosome-loaded dECM hydrogels on acute liver failure (ALF), a series of experiments were conducted. H&E staining and TUNEL staining results showed ( Figure 4 In Figures a and b), compared to the control group (ALF) and the exosome group (exos), the dECM hydrogel group loaded with exosomes (E-exos) showed the least periportal necrosis and the least green fluorescence, representing apoptosis, indicating that the combined use of exosomes and dECM hydrogels has a synergistic protective effect in the ALF model. Seven-day survival monitoring further showed that the E-exos group had the highest survival rate.Figure 4 In addition, serum biochemical analysis showed (Fig. 6c) that the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the E-exos group were significantly lower than those in the ALF group and the exos group, indicating that the exosome-loaded dECM hydrogel could effectively alleviate liver cell damage and promote liver function recovery. Figure 4

[0037] Example 5: Functionality of exosome-loaded dECM hydrogel in liver repair To explore the specific role of exosome-loaded dECM hydrogel in liver repair, a series of experiments were carried out. The anti-inflammatory performance was evaluated by detecting the expression of inflammation-related proteins and genes in liver tissue. The results showed that, at the protein level, the expression of IL-6 and TNF-a proteins in the E-exos group was significantly lower than that in the acute liver failure group ALF group and the exos group (Fig. 6a and b), indicating that the exosome-loaded dECM hydrogel had clear anti-inflammatory properties; this was further confirmed at the gene level (Fig. 6c): the mRNA expression of TNF-a, IL-6 and COX2 in the untreated ALF group was significantly up-regulated; while exos treatment, especially the E-exos group, could significantly inhibit the expression of these inflammatory genes. The above results showed that the exosome-loaded dECM hydrogel could effectively regulate the inflammatory response during liver injury. Figure 5 Figure 5

[0038] The above are only preferred embodiments of the present application, and it should be pointed out that for those skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which should be considered as the protection scope of the present application, and these will not affect the effect and practicality of the present application.​​​

Claims

1. A bio-mixed decellularized liver cancer adjacent tissue hydrogel, characterized in that, Including decellularized hydrogels and mesenchymal stem cell-derived exosomes, per 1 cm 3 A decellularized hydrogel was loaded with 1 mg of mesenchymal stem cell-derived exosomes; the decellularized hydrogel was formed from human liver cancer adjacent tissue after decellularization and enzymatic digestion; the mesenchymal stem cell-derived exosomes were obtained from the mesenchymal stem cell culture supernatant by differential centrifugation.

2. The bio-mixed decellularized liver cancer adjacent tissue hydrogel according to claim 1, characterized in that, The preparation steps of the decellularized hydrogel are as follows: Human liver cancer adjacent tissue blocks were taken and immersed in a solution containing 1% Triton-X 100 and incubated at 4°C. The tissue was repeatedly washed with sterile PBS to complete the decellularization process and obtain decellularized extracellular matrix. The decellularized extracellular matrix was placed in a centrifuge tube containing digestion solution and incubated in a 37°C water bath for 2 hours. Subsequently, the digested solution was filtered through a cell filter, and the filtered digestion solution was incubated at 37°C for 1 hour to form a decellularized hydrogel. The digestive fluid consists of 0.25% trypsin and 0.05% type II collagenase.

3. The bio-mixed decellularized liver cancer adjacent tissue hydrogel according to claim 1, characterized in that, The extraction steps for the mesenchymal stem cell-derived exosomes are as follows: Rat bone marrow fluid was collected and centrifuged at 300-400g for 5 min, then the supernatant was discarded. The bone marrow cell pellet was resuspended in serum-free DMEM basal medium, and then 10% fetal bovine serum and 1% penicillin-streptomycin were added to the medium to prepare a complete mesenchymal stem cell culture medium. The cells were seeded into sterile culture dishes and cultured at 37℃ and 5% CO2. When the cells reached 90% confluence, the complete mesenchymal stem cell culture medium was collected first, and then 0.25% trypsin was added to the culture dish to digest the cells. The cells were then separated from the culture dish and passaged. The collected mesenchymal stem cell culture medium was centrifuged at 400g for 10 min, and the supernatant was transferred to a sterile centrifuge tube and centrifuged at 2000g for 10-20 min. The supernatant was centrifuged at 10000-20000g for 30 min, and the pellet containing exosome particles was collected and resuspended in PBS.

4. A three-dimensional hepatocyte culture system, characterized in that, This includes the bio-mixed decellularized hepatocellular carcinoma adjacent tissue hydrogel as described in any one of claims 1-3 and hepatocytes derived from human induced pluripotent stem cells.

5. The three-dimensional hepatocyte culture system according to claim 4, characterized in that, The three-dimensional hepatocyte culture system is used to promote hepatocyte proliferation or maintain hepatocyte function in vitro.

6. The three-dimensional hepatocyte culture system according to claim 4, characterized in that, The three-dimensional hepatocyte culture system is used to establish an in vitro liver injury model.

7. The three-dimensional hepatocyte culture system according to claim 4, characterized in that, The steps for constructing the three-dimensional hepatocyte culture system are as follows: Hepatocytes derived from human induced pluripotent stem cells were co-cultured with a culture medium supplemented with bio-mixed decellularized adjacent tissue hydrogel of hepatocellular carcinoma at 37°C and 5% CO2 to obtain a three-dimensional hepatocyte culture system. The culture medium is DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin.

8. The three-dimensional hepatocyte culture system according to claim 7, characterized in that, In the aforementioned three-dimensional hepatocyte culture system, each 1cm 3 Cell-free hydrogel loaded with 1×10 7 Hepatocytes derived from personally induced pluripotent stem cells.

9. The use of a bio-mixed decellularized hepatocellular carcinoma adjacent tissue hydrogel according to any one of claims 1-3 in the preparation of formulations for in vitro culture of hepatocytes or establishment of in vitro liver injury models.

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