Biological mixed decellularized liver cancer paracancerous tissue hydrogel, preparation method therefor, and use thereof
Patent Information
- Application Number
- PCT/CN2025/135769
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2025-11-07
- Filing Date
- 2025-11-18
- Publication Date
- 2026-10-22
Smart Images
Figure CN2025135769_22102026_PF_FP_ABST
Abstract
Description
A bio-mixed decellularized liver cancer adjacent tissue hydrogel, its preparation method and application Technical Field
[0001] This invention belongs to the field of biomedical materials technology, and particularly relates to a bio-mixed decellularized liver cancer adjacent tissue hydrogel, its preparation method and application. Background Technology
[0002] Acute liver failure (ALF) is a serious clinical disease characterized by rapid progression and high mortality, primarily marked by a sharp decline in liver function and extensive hepatocyte necrosis. Currently, liver transplantation is the primary treatment for ALF; however, the severe shortage of donor organs and the various risks associated with the transplantation process significantly limit its widespread application. Against this backdrop, tissue engineering techniques are gradually emerging as a promising alternative strategy for treating ALF. Among these, decellularized scaffolds, by removing cellular components while retaining the natural extracellular matrix (ECM), provide an ideal platform for liver tissue regeneration. These scaffolds not only support cell adhesion and proliferation but are also rich in bioactive molecules that guide cell behavior and tissue structure.
[0003] Tissue samples obtained clinically are often considered medical waste, but they can actually serve as potential sources of natural scaffolds. In particular, tumor peritumoral tissue that has undergone mild decellularization can retain a large number of bioactive components while maintaining low immunogenicity, showing promising clinical application prospects.
[0004] Mesenchymal stem cells (MSCs), as a type of adult stem cell with self-renewal and multi-lineage differentiation potential, play an important role in tissue repair. Integrating MSC-derived exosomes into a hydrogel system can effectively enrich their endogenous regenerative cytokines. These exosomes carry a variety of bioactive substances that can mediate tissue repair, regulate inflammatory responses, and promote cell survival through paracrine mechanisms.
[0005] Despite the potential of tissue engineering in the treatment of ALF, current treatment strategies still have several significant limitations. On the one hand, the scarcity of autologous decellularized scaffolds limits their widespread application; on the other hand, scaffolds simply inoculated with hepatocytes lack efficient regeneration-promoting mechanisms and sufficient anti-inflammatory capabilities, thus affecting treatment efficacy. Therefore, developing novel treatment strategies to improve the survival rate of ALF patients and restore liver function is a crucial issue that urgently needs to be addressed.
[0006] Therefore, this invention proposes a bio-mixed decellularized liver cancer adjacent tissue hydrogel, its preparation method, and its application. Summary of the Invention
[0007] The purpose of this invention is to provide a bio-mixed decellularized liver cancer adjacent tissue hydrogel, its preparation method and application, in order to solve the problems mentioned in the background art.
[0008] The objective of this invention is achieved through the following technical solution:
[0009] A bio-mixed decellularized hepatocellular carcinoma adjacent tissue hydrogel, comprising decellularized hydrogel 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.
[0010] Furthermore, the preparation steps of the decellularized hydrogel are as follows:
[0011] 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.
[0012] The digestive fluid consists of 0.25% trypsin and 0.05% type II collagenase.
[0013] Furthermore, the extraction steps for the mesenchymal stem cell-derived exosomes are as follows:
[0014] 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.
[0015] A three-dimensional hepatocyte culture system comprising, as described above, a bio-mixed decellularized hepatocellular carcinoma adjacent tissue hydrogel and hepatocytes derived from human induced pluripotent stem cells.
[0016] Furthermore, the three-dimensional hepatocyte culture system is used to promote hepatocyte proliferation or maintain hepatocyte function in vitro.
[0017] Furthermore, the three-dimensional hepatocyte culture system is used to establish an in vitro liver injury model.
[0018] Furthermore, the construction steps of the three-dimensional hepatocyte culture system are as follows:
[0019] 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.
[0020] The culture medium is DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin.
[0021] Furthermore, in the aforementioned three-dimensional hepatocyte culture system, each 1 cm 3 Cell-free hydrogel loaded with 1×10 7 Hepatocytes derived from personally induced pluripotent stem cells.
[0022] The application of a bio-mixed decellularized hepatocellular carcinoma adjacent tissue hydrogel as described above in the preparation of formulations for in vitro culture of hepatocytes or establishment of in vitro liver injury models.
[0023] Compared with the prior art, the beneficial effects of the present invention are:
[0024] 1. The decellularized hydrogel prepared by this invention has a wide range of raw material sources, and it can transform clinically available waste (such as human liver cancer adjacent tissue blocks) into scaffold materials, effectively solving the problem of scaffold scarcity and realizing the resource utilization of clinical waste.
[0025] 2. The decellularized hydrogel prepared by this invention completely retains the natural extracellular matrix, exhibiting not only low immunogenicity but also excellent biocompatibility. This hydrogel can provide a highly biomimetic structural platform for cells (such as hepatocytes derived from human induced pluripotent stem cells), fully meeting the needs of cell adhesion, growth, and proliferation.
[0026] 3. The decellularized hydrogel prepared by this invention can effectively integrate exogenous regenerative active substances (such as exosomes derived from mesenchymal stem cells), and through the synergistic effect of the two, it can significantly enhance the ability to regulate inflammation, promote tissue repair and improve cell survival, providing a novel and effective treatment for acute liver failure. Attached Figure Description
[0027] Figure 1 is a schematic diagram of the construction of the iPSC-Heps three-dimensional co-culture system based on decellularized hydrogel loaded with exosomes.
[0028] Figure 2 shows the characterization of the decellularized hydrogel; where: a is the degradation experiment; b is the erosion experiment; c is the swelling experiment; and d is the viscoelasticity experiment.
[0029] Figure 3 shows the biocompatibility of decellularized hydrogels and decellularized hydrogels loaded with exosomes; where: a is live / dead staining; b is the CCK8 assay.
[0030] Figure 4 shows the therapeutic effect of decellularized hydrogel loaded with exosomes; where: a is H&E staining; b is TUNEL staining; c is survival rate; d is liver function indicators (ALT, AST).
[0031] Figure 5 shows the liver repair effect of decellularized hydrogels loaded with exosomes; where: a is immunofluorescence (TNF-α); b is immunofluorescence (IL-6); c is the gene expression levels of TNF-α, IL-6, and COX2. Detailed Implementation
[0032] To provide a clearer understanding of the technical features, objectives, and beneficial effects of this invention, the technical solution of this invention is described in detail below, but this should not be construed as limiting the scope of implementation of this invention. Unless otherwise specified, the methods used in this invention are conventional methods in this technical field. In this invention, materials, reagents, or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.
[0033] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.
[0034] Example 1: Construction of a three-dimensional co-culture system of iPSC-Heps based on dECM hydrogel loaded with exosomes (Figure 1);
[0035] Step 1: Preparation of decellularized (dECM) hydrogel;
[0036] (1) Take a block of tissue adjacent to human liver cancer, immerse it in a solution containing 1% Triton-X 100, and incubate it at 4°C to destroy the cell membrane of the cells in the tissue and release the cell contents;
[0037] (2) Rinse the above tissue repeatedly (at least three times) with a large amount of sterile PBS to remove residual detergent, cell debris and dissolved substances, and complete the decellularization process to obtain decellularized extracellular matrix.
[0038] (3) Place the decellularized extracellular matrix into a centrifuge tube containing digestion solution (composed of 0.25% trypsin and 0.05% type II collagenase) and incubate in a 37°C water bath for 2 hours; then filter the digested solution through a 100μm cell filter to remove undigested tissue blocks and large particles.
[0039] (4) The filtered digestion solution was incubated at 37°C for 1 hour to form a decellularized (dECM) hydrogel.
[0040] Step 2: Extraction of exosomes derived from mesenchymal stem cells;
[0041] (1) Bone marrow fluid was collected from rats (purchased from Spiford (Suzhou) Biotechnology Co., Ltd.), centrifuged at low speed (approximately 300g, for 5 minutes), and the supernatant was discarded to remove 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 (PS) 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°C and 5% CO2. Once the cells reached 90% confluence, the complete mesenchymal stem cell culture medium was collected, 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.
[0042] (2) Centrifuge the collected mesenchymal stem cell culture medium at 400g for 10min to remove suspended cells and cell debris; transfer the supernatant to a sterile centrifuge tube and centrifuge at 2000g for 20min to remove larger particulate contaminants.
[0043] (3) Centrifuge the supernatant at 20000g for 30min, collect the precipitate containing exosome particles and resuspend it in PBS.
[0044] Step 3: Construction of a three-dimensional co-culture system of iPSC-Heps based on dECM hydrogel loaded with exosomes;
[0045] Human induced pluripotent stem cell-derived hepatocytes (iPSC-Heps) were co-cultured in DMEM medium supplemented with dECM hydrogel prepared in step 1 and exosomes obtained in step 2, containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS) at 37°C and 5% CO2 to construct a three-dimensional (3D) co-culture system. In this system, each 1 cm 3 dECM hydrogel loading 1×10 7 One iPSC-Heps cell and 1 mg of exosomes.
[0046] Example 2: In vitro stability characterization of dECM hydrogel;
[0047] To investigate the effect of enzymes on hydrogel degradation, dECM hydrogels were immersed in PBS containing 5 U / mL and 10 U / mL type II collagenase, respectively, and degradation experiments were conducted at 37°C. With increasing incubation time in the type II collagenase solution, the dECM hydrogels 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 (Figure 2a). To investigate the effect of pH on dECM hydrogel erosion, the erosion of dECM hydrogels was observed under pH=5.5 and pH=7.4 conditions. Figure 2b shows that the erosion rate of dECM hydrogels was relatively slow under both pH conditions, indicating that dECM hydrogels maintain stability in the absence of enzymes. Swelling assays showed that dECM hydrogels exhibited rapid swelling behavior within 6 hours, with a swelling rate approaching 1200% (Figure 2c). Furthermore, the viscoelasticity of the dECM hydrogel was analyzed. The G' and G'' values remained relatively stable within the strain range of 1% to 10%, indicating that the dECM hydrogel has a linear viscoelastic region (d in Figure 2).
[0048] Example 3: Biocompatibility of dECM hydrogels and dECM hydrogels loaded with exosomes;
[0049] 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) methods, and cell viability was assessed. 2D culture (dECM hydrogel group): iPSC-Heps were seeded on 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 (Figure 3a) showed that when iPSC-Heps were cultured on the surface of dECM hydrogels for up to 3 days, green fluorescence (live cells) was continuously and densely distributed, indicating good cell viability. CCK8 assay results (Figure 3b) showed that the cell viability of the 3D culture group was higher than that of the 2D culture group. The above results indicate that dECM hydrogels exhibit low toxicity to iPSC-Heps and excellent biocompatibility. Furthermore, compared to 2D culture, exosome-loaded dECM hydrogels can further promote cell proliferation in 3D culture, demonstrating the potential of exosome-loaded dECM hydrogels in promoting cell growth.
[0050] Example 4: The therapeutic effect of exosome-loaded dECM hydrogel on acute liver failure;
[0051] To investigate the therapeutic effect of exosome-loaded dECM hydrogels on acute liver failure (ALF), a series of experiments were conducted. H&E and TUNEL staining results (Figure 4a and b) showed that, compared with the control group (ALF) and the exosome group (exos), the exosome-loaded dECM hydrogel group (E-exos) had the least periportal necrosis area and the least green fluorescence representing apoptosis, indicating that the combined use of exosomes and dECM hydrogels had 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 4c). In addition, serum biochemical analysis (Figure 4d) showed that the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the E-exos group were significantly lower than those in the ALF and exos groups, indicating that exosome-loaded dECM hydrogels can effectively reduce hepatocyte damage and promote liver function recovery.
[0052] Example 5: Functional investigation of exosome-loaded dECM hydrogel in liver repair;
[0053] To investigate the specific role of exosome-loaded dECM hydrogels in liver repair, a series of experiments were conducted. The anti-inflammatory properties were assessed by detecting the expression of inflammation-related proteins and genes in liver tissue. Results showed that at the protein level, the expression of IL-6 and TNF-α proteins in the E-exos group was significantly lower than that in the acute liver failure group (ALF group) and the exos group (Figure 5a and b), suggesting that the exosome-loaded dECM hydrogels possess clear anti-inflammatory properties. Further confirmation at the gene level (Figure 5c) indicated that the mRNA expression of TNF-α, IL-6, and COX2 was significantly upregulated in the untreated ALF group; while exos treatment, especially the E-exos group, significantly inhibited the expression of these inflammatory genes. These results demonstrate that exosome-loaded dECM hydrogels can effectively regulate the inflammatory response during liver injury.
[0054] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent.
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 construction steps of 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.