A bionic micro liver tissue and its preparation method and application

Abstract

The present invention relates to the field of biotechnology, and in particular to a bionic micro-liver tissue and a preparation method and application thereof. Compared with the conventional strategy of cell differentiation followed by co-culture, the present invention directly induces differentiation of mesenchymal stem cell spheres in co-culture, which not only improves the liver differentiation efficiency of mesenchymal stem cells, but also simplifies the operation steps. At the same time, ectopic subcutaneous implantation of the micro-liver tissue of the present invention also shows a significant therapeutic effect for liver failure.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a bionic micro-liver tissue and a preparation method and application thereof. Background Art

[0002] The hepatocyte-like cells induced to differentiate into by stem cells in a two-dimensional culture dish are often not mature enough and have difficulty in exerting normal liver function, thus limiting their efficacy in treating acute liver failure. Cell spheres formed by multicellular aggregation have a higher potential for liver differentiation, but their size effect has rarely been reported. The present invention achieves efficient liver differentiation by controlling the size of mesenchymal stem cell spheres. In addition, vascularization is also the key to the timely efficacy of artificial liver tissue. Conventional methods of simply mixing vascular endothelial cells with differentiated hepatocyte-like cells cannot well simulate the specific vascular structure in the liver lobule. Based on this, the present invention also uses 3D coaxially printed vascular endothelial cells to construct a biomimetic vascular structure of the liver lobule, and by co-culturing with mesenchymal stem cell spheres, the efficiency of liver differentiation of mesenchymal stem cells is further improved. The constructed vascularized micro-liver tissue significantly promoted liver regeneration in mice with acute liver failure after subcutaneous transplantation.

[0003] Mesenchymal stem cells are usually differentiated into hepatocyte-like cells in conventional two-dimensional culture dishes by induction culture medium. In order to improve the efficiency of hepatic differentiation of mesenchymal stem cells, they are often embedded in three-dimensional hydrogels or engineered into multicellular cell spheres to better simulate the microenvironment of cells in the body. In addition, hepatocyte function can also be improved by co-culturing hepatocyte-like cells and vascular endothelial cells, among which the proportion and spatial distribution of vascular endothelial cells are important influencing factors. The hepatic lobule is the smallest functional unit of liver tissue and has a hexagonal interconnected vascular network. Existing technologies mainly use preset extrusion bioprinting or digital light processing to alternate the distribution of hepatocytes and vascular endothelial cells to simply simulate the hexagonal structure of the hepatic lobule.

[0004] While spheroids formed by multicellular aggregation have higher hepatic differentiation potential than the same number of single cells, spheroid size is highly correlated with function, and existing technologies rarely address this size effect, resulting in inconsistent efficacy. Furthermore, most existing technologies pre-induce hepatic differentiation of mesenchymal stem cells before co-culturing them with vascular endothelial cells, resulting in complex procedures. Furthermore, while pre-extrusion bioprinting and digital light processing can construct biomimetic liver lobule structures, their vascular endothelial cells are simply distributed in a hexagonal structure alternating with hepatocytes, lacking complete connectivity and hollow vascular structures. Summary of the Invention

[0005] In light of this, the present invention provides a biomimetic micro-liver tissue, its preparation method, and application. The biomimetic liver lobule constructed in this invention features a hexagonal interconnected vascular network. By co-culturing with mesenchymal stem cell spheres and inducing their hepatic differentiation, the micro-liver tissue was successfully produced, demonstrating therapeutic effects in treating acute liver failure.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for preparing a bionic liver lobule-like vascular structure, comprising:

[0008] Step 1) preparing an outer layer supporting ink and an inner layer sacrificial ink respectively;

[0009] Step 2) performing 3D coaxial bioprinting using the outer support ink and inner sacrificial ink according to a preset hexagonal pattern with a central circular hole and twelve equal radial sections, thereby obtaining a hexagonal structure;

[0010] Step 3) placing the hexagonal structure in a cross-linking solution to cross-link and shape the outer supporting ink, removing the inner sacrificial ink, and obtaining a bionic liver lobule-like vascular structure.

[0011] In step 1) of the present invention, the outer supporting ink comprises vascular endothelial cells and the following components by mass fraction: 0.1-2% sodium alginate, 1-8% gelatin and 0.1-10% fibrinogen; the inner sacrificial ink comprises 1-8% gelatin, 0.1-2% sodium hyaluronate and 1-100 units / ml thrombin.

[0012] In some specific embodiments, the outer supporting ink includes vascular endothelial cells, 1.5% sodium alginate, 5% gelatin and 0.1% fibrinogen; the inner sacrificial ink includes 7.5% gelatin, 1.5% sodium hyaluronate and 8 units / ml thrombin.

[0013] In the present invention, in step 2), the printing conditions are:

[0014] The extrusion pressures of the supporting ink and sacrificial ink are 0.01-0.1 MPa and 0.1-0.5 MPa, respectively, and the temperature is 15-25°C. The diameter of the needle for the inner layer printing is 0.1-1 mm, and the diameter of the needle for the outer layer printing is 0.5-5 mm, both at a temperature of 15-20°C. The inner layer printing needle has a smaller diameter than the outer layer printing needle, and the temperatures of the inner and outer needles are lower than the ink temperature.

[0015] In some specific embodiments, the printing conditions are:

[0016] The extrusion pressures of the supporting ink and the sacrificial ink are 0.05 MPa and 0.35 MPa respectively, and the temperature is 25°C; the diameter of the needle for printing the inner layer is 0.26 mm, the diameter of the needle for printing the outer layer is 0.8 mm, and the needle temperature is 20°C.

[0017] In the present invention, in step 3), the cross-linking solution comprises 40 mmol / L calcium chloride, 0.9 wt% sodium chloride, and 8 units / mL thrombin; the cross-linking solution is a solution pre-cooled at 4° C., and the cross-linking time is 30 min;

[0018] The method for removing the inner layer of sacrificial ink includes: immersing the cross-linked structure in a phosphate buffer solution preheated at 37°C.

[0019] The present invention also provides a bionic liver lobule vascular structure prepared by the preparation method.

[0020] The biomimetic liver lobule vascular structure printed in the present invention is a hexagonal structure with hollow circular tubes resembling a liver lobule. This structure comprises equally divided hepatic sinusoidal regions. The number of divisions can be determined by those skilled in the art based on actual conditions. In the present invention, the divisions can range from 6 to 96, specifically 6, 12, 24, 48, or 96.

[0021] The present invention also provides a method for preparing a bionic micro-liver tissue, comprising:

[0022] Resuspending vascular endothelial cells and stem cell spheres in the same hydrogel to obtain a mixture;

[0023] adding the mixture into the gaps of the biomimetic liver lobule-like vascular structure, incubating, and adding liver differentiation medium after the hydrogel is gelled to induce hepatic differentiation of mesenchymal stem cells to obtain biomimetic micro-liver tissue;

[0024] The stem cell spheres include one or more of mesenchymal stem cell spheres, embryonic stem cell spheres, induced pluripotent stem cell spheres and hepatoblast spheres.

[0025] In some specific embodiments of the present invention, the stem cell spheres are mesenchymal stem cell spheres. Specifically, the mesenchymal stem cell spheres are obtained by microwell culture, and can also be prepared by other methods, such as low-adsorption culture dish culture, Aggrewell culture of STEMCELL Technologies Inc. TM Product culture, multi-well culture dish culture, hanging drop culture, microsphere culture made by microfluidic technology, dynamic culture in stirred bioreactor, etc.

[0026] The present invention obtains mesenchymal stem cell spheres of different sizes by controlling the cell inoculation amount and using micropores of different specifications. In some embodiments, the diameter of the stem cell sphere is 50 to 150 μm, specifically 50 μm, 100 μm, and 150 μm. The present invention compares the effects of various cell sphere sizes (50 μm, 100 μm, 150 μm, and 200 μm) within the oxygen uptake limit distance (200 microns) on their hepatotropic differentiation, and determines that a diameter of 50 microns is the optimal size, which can maximize the hepatotropic differentiation efficiency of mesenchymal stem cell spheres.

[0027] In the present invention, the cell number ratio of the vascular endothelial cells and stem cell spheres is (0-4):1; in some specific embodiments, the ratio is specifically 2:1, 3:1, or 4:1. In a specific embodiment, the present invention co-cultures vascular endothelial cells and mesenchymal stem cell spheres (50 microns in diameter) at different ratios (0:1, 1:1, 2:1, and 4:1) in a liver decellularized matrix hydrogel and induces hepatic differentiation of mesenchymal stem cells. The expression differences of liver function-related genes at the (a) mRNA and (b) protein levels were compared. The results showed that a 2:1 co-culture ratio of vascular endothelial cells and mesenchymal stem cell spheres significantly promoted the hepatic differentiation of mesenchymal stem cells.

[0028] In the present invention, the bionic liver lobule-like vascular structure is a liver lobule-like vascular structure prepared by the above preparation method of the present invention, and has a hexagonal hollow interconnected vascular structure.

[0029] In the present invention, the vascular endothelial cells include: endothelial cells derived from umbilical vein, hepatic sinusoid, artery, dermal microvessels or vascular endothelial cells differentiated from stem cells. In some specific embodiments, the vascular endothelial cells are human umbilical vein endothelial cells.

[0030] In the present invention, the incubation time is 37° C. for 1 hour, and the induction time for hepatic differentiation is 23 days.

[0031] In the present invention, the hydrogel is a liver decellularized matrix hydrogel, or a hydrogel made from the following materials: gelatin, hyaluronic acid, polyethylene glycol, and its derivatives. In some specific embodiments, the hydrogel is a liver decellularized matrix hydrogel. A 1-cubic-centimeter piece of pig liver is prepared by freezing and thawing three times between -80°C and room temperature, washing with ultrapure water to remove blood cells. The liver is then incubated in a decellularized composite solution of 1% Triton X-100 / 0.1% ammonia for three days with continuous stirring, with the water changed three times daily. Finally, the solution is freeze-dried, ground into a powder, weighed, and digested in 0.1 M sodium hydroxide solution at a weight ratio of 10:1 liver decellularized extracellular matrix to pepsin for 36 hours. The liver decellularized matrix hydrogel is used at a concentration of 10 mg / mL and a pH of 7.2.

[0032] The present invention also provides bionic micro-liver tissue prepared by the above preparation method.

[0033] The bionic micro liver tissue prepared by the present invention has a diameter of 100 microns to 1 centimeter and a height of 100 microns to 1 centimeter, and can contain 6, 12, 24, 48 or 96 equally divided liver sinusoidal areas.

[0034] The present invention also provides a method for treating acute liver failure, comprising: implanting the micro-liver tissue described herein. Implantation sites include the subcutaneous, vascular-rich fat pad in the groin, the mesentery, the liver capsule, lymph nodes, and the like. Acute liver failure includes liver failure induced by carbon tetrachloride, acetaminophen, D-galactosidase / lipopolysaccharide, or liver failure caused by hepatic ischemia-reperfusion.

[0035] In the present invention, the implanted micro-liver tissue is modular in design and can be stacked in one or more ways, wherein the number of hepatocyte-like cells contained in each micro-liver tissue is 1×10 6 -1×10 9 indivual.

[0036] The present invention also provides the use of the bionic micro-liver tissue of the present invention in the safety evaluation of hepatotoxic drugs or cosmetics. The hepatotoxic drugs include traditional Chinese medicine formulas, Chinese patent medicines or other Western medicines containing hepatotoxic substances.

[0037] The present invention used 3D coaxial bioprinting of vascular endothelial cells to construct a hexagonally interconnected vascular network reminiscent of a liver lobule. This network was then co-cultured with mesenchymal stem cell spheres, directly inducing hepatic differentiation of the mesenchymal stem cells during the co-culture process. This successfully produced micro-liver tissues with therapeutic effects on acute liver failure. This not only improved the efficiency of hepatic differentiation of the mesenchymal stem cells but also simplified the procedure. Experimental results demonstrated that the micro-liver tissues constructed by the present invention successfully promoted liver repair and regeneration in mice with acute liver failure induced by carbon tetrachloride and acetaminophen. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Flow chart showing the construction of micro-liver tissue;

[0039] Figure 2 Figure 3. Mesenchymal stem cell spheres of three sizes prepared from single cells and micropores: S spheres (diameter: 50 μm), M spheres (diameter: 100 μm), and L spheres (diameter: 150 μm). a represents cell viability (scale bar: 100 μm), bf represent hepatic differentiation, b-c represent mRNA expression levels of genes related to liver function, and e-f represent protein levels (scale bar: 50 μm).

[0040] Figure 3Figure 3 shows the (a) morphology (large graph scale: 200 μm; small graph scale: 50 μm) and (bc) hepatic differentiation (expression of liver function-related genes at the mRNA and protein levels) of three sizes of mesenchymal stem cell spheres (XS spheres (diameter: 25 μm), S spheres (diameter: 50 μm), and XL spheres (diameter: 200 μm)) prepared using microwells.

[0041] Figure 4 Figure 3 shows a 3D coaxially printed biomimetic liver lobule-like vasculature and a thermosensitive liver decellularized matrix hydrogel; (b) Scanning electron microscopy reveals a hollow tubular structure, scale bar: 200 μm; (c) Live-dead staining demonstrates that the 3D-printed vascular endothelial cells have good cell viability and that the biomimetic liver lobule maintains structural stability in culture medium, scale bar: 200 μm; (d) Brightfield microscopy of vascular endothelial cells embedded solely in a liver decellularized matrix hydrogel, vascular endothelial cells and mesenchymal stem cell spheres co-cultured in a liver decellularized matrix hydrogel, and vascular endothelial cells and mesenchymal stem cell spheres filling the gap between 3D-printed blood vessels and liver sinusoids, scale bar: 500 μm.

[0042] Figure 5 Figure 3. Co-culture of vascular endothelial cells and 50-μm-diameter mesenchymal stem cell spheres at different ratios (0:1, 1:1, 2:1, and 4:1) in liver decellularized matrix hydrogels to induce hepatic differentiation of mesenchymal stem cells. (a) Expression differences of liver function-related genes at the mRNA and protein levels (b). Scale bar: 50 μm.

[0043] Figure 6 Figure 1 shows the expression differences of liver function-related genes at the (a) mRNA and (b) protein levels in comparative examples 1-2, where randomly or hexagonally distributed vascular endothelial cells and mesenchymal stem cell spheres were mixed at a ratio of 2:1 in a hydrogel containing a liver decellularized matrix and co-cultured to induce hepatic differentiation of mesenchymal stem cells. Scale bar: 100 μm.

[0044] Figure 7 Comparative Examples 3-4: Vascular endothelial cells and mesenchymal stem cell spheres were randomly mixed at a ratio of 2:1 and co-cultured in liver decellularized matrix hydrogels with and without hexagonal hollow channels. Mesenchymal stem cells were induced to differentiate into hepatic endogenous genes. Expression differences of liver function-related genes at the (a) mRNA and (b) protein levels are shown. Scale bar: 100 μm.

[0045] Figure 8(a) Liver tissue morphology and abnormal area statistics of different treatment groups, scale bar: 100 μm; (c) Oxidative stress levels of liver tissues of different treatment groups; (d) Differences in the expression of inflammatory factor genes (Nos2 and Tnfa) at the mRNA level in liver tissues of different treatment groups; (e) Immunofluorescence staining of cyclooxygenase-2 expression levels in liver tissues of different treatment groups, scale bar: 200 μm; (f) Enzyme-linked immunosorbent assay of tumor necrosis factor TNF-α, inducible nitric oxide synthase iNOS, and anti-inflammatory factor (interleukin-10) in liver tissues of different treatment groups;

[0046] Figure 9 (a) Liver tissue morphology of different treatment groups; (b) Differences in the mRNA expression of inflammatory factor genes (Tnfa, Il1b, Nos2, and Il6), antioxidant genes (Sod1 and Sod2), and liver function-related genes (Alb and Cyp3a11) in the liver tissues of different treatment groups; (c) Immunoblotting to detect the levels of tumor necrosis factor TNF-α and inducible nitric oxide synthase iNOS relative to the internal reference protein GAPDH in the liver tissues of different treatment groups; (d) Enzyme-linked immunosorbent assay to determine the content of tumor necrosis factor TNF-α in the liver tissues of different treatment groups; (e) Immunofluorescence staining to detect the expression levels of granulocyte markers (lymphocyte antigen-6G) and cell proliferation marker Ki-67 in the liver tissues of different treatment groups, scale bar: 200 μm. DETAILED DESCRIPTION

[0047] The present invention provides a biomimetic micro-liver tissue, its preparation method, and application. Those skilled in the art may refer to the contents herein and appropriately modify the process parameters to achieve the desired results. It should be noted that all similar substitutions and modifications obvious to those skilled in the art are considered encompassed by the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel will clearly be able to modify or appropriately alter and combine the methods and applications herein to implement and apply the technology of the present invention without departing from the content, spirit, and scope of the present invention.

[0048] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0049] The present invention will be further described below in conjunction with the embodiments:

[0050] Example 1 Preparation of Mesenchymal Stem Cell Spheres

[0051] Different seeding amounts of mesenchymal stem cells (10k, 25k and 50k) were seeded into microwell arrays of the same size (length × width × height: 0.5 mm × 0.5 mm × 0.22 mm, a total of 100 microwells), and after centrifugation at 600 rcf for 5 minutes and overnight culture, mesenchymal stem cell spheres with diameters of 50 μm (S cell sphere), 100 μm (M cell sphere) and 150 μm (L cell sphere) were obtained in turn. Figure 2 ab). 10k means 10,000 cells were seeded per microwell array.

[0052] The mRNA and protein expression levels of liver function-related genes in single cells and the above-mentioned mesenchymal stem cell spheres were detected. The results showed that compared with single cells cultured in two-dimensional culture dishes (single cells), mesenchymal stem cell spheres had better liver differentiation potential (expression of liver-related genes at the transcriptional and translational levels), and the differentiation efficiency of cell spheres with a diameter of 50 microns (S cell spheres) was higher ( Figure 2 c~f).

[0053] At the same time, the same number of cells (10k) were seeded into microwell arrays of different sizes (length × width × height: 0.33 mm × 0.33 mm × 0.3 mm, a total of 225 microwells; length × width × height: 0.5 mm × 0.5 mm × 0.22 mm, a total of 100 microwells; length × width × height: 1.3 mm × 1.3 mm × 0.9 mm, a total of 10 microwells), centrifuged at 600 rcf for 5 minutes and then cultured overnight to obtain mesenchymal stem cell spheres with diameters of 25 μm (XS cell sphere), 50 μm (S cell sphere) and 200 μm (XL cell sphere) ( Figure 3 a). The results of liver-related gene mRNA and protein expression levels showed that the cell spheres with a diameter of 50 microns (S cell spheres) had the highest liver differentiation ability ( Figure 3 bc).

[0054] Example 2 3D coaxial bioprinting biomimetic liver lobule vascular structure

[0055] 1) Preparation of outer support ink and inner sacrificial ink: The outer support ink (ESI) consists of 1.5% sodium alginate, 5% gelatin, 0.1% fibrinogen and vascular endothelial cells, and the inner sacrificial ink (ISI) mainly consists of 7.5% gelatin, 1.5% sodium hyaluronate and 8 units / ml thrombin.

[0056] 2) The inner and outer needles have diameters of 0.26 mm and 0.8 mm, respectively, and are maintained at 20°C. The inner and outer sacrificial inks are extruded at 25°C using air pressures of 0.05 MPa and 0.35 MPa, respectively, onto a receiving plate temperature-controlled at 4°C. The printed pattern is a twelve-equal-divided hexagonal hollow circular hole structure.

[0057] 3) The printed structure was immersed in a cross-linking solution (40 mmol / L calcium chloride, 0.9% sodium chloride, and 8 units / ml thrombin) pre-cooled at 4 degrees Celsius for 30 minutes to allow the outer support ink to gel. Finally, the formed structure was immersed in a 1× phosphate buffer solution pre-heated at 37 degrees Celsius to remove the inner sacrificial ink, thereby forming an empty tube structure and obtaining a biomimetic liver lobule vascular structure (see Figure 4 ).

[0058] Example 3 Preparation of bionic micro-liver tissue of the present invention

[0059] Endothelial cells and mesenchymal stem cell spheres with a diameter of 50 μm prepared in Example 1 were embedded in liver decellularized matrix hydrogel at different cell density ratios (0:1, 1:1, 2:1 and 4:1). The seeding density of mesenchymal stem cells was 5×10 6 Cells / ml were mixed into the gaps of the 3D printed biomimetic liver lobule vascular structure of Example 2 and incubated at 37 degrees Celsius for 1 hour. After the acellular matrix hydrogel was gelled, an equal volume of liver differentiation medium was added to start inducing hepatic differentiation of mesenchymal stem cells. After 23 days of culture (i.e., co-culture process), micro liver tissue was obtained (see Figure 5 The density of vascular endothelial cells in the 3D-printed biomimetic liver lobule vascular structure was consistent with the density of vascular endothelial cells mixed in the acellular matrix hydrogel.

[0060] Example 4 Preparation of Micro-liver Tissues by Co-culturing Vascular Endothelial Cells of Different Morphologies with Mesenchymal Stem Cells

[0061] (1) Preparation of micro-liver tissue in control group 1 (compared with Example 3, this control group did not include a 3D-printed vascular structure)

[0062] Randomly distributed vascular endothelial cells and 50 μm diameter mesenchymal stem cell spheres prepared in Example 1 were mixed at a ratio of 2:1, embedded in liver decellularized matrix hydrogel, and incubated at 37 degrees Celsius for 1 hour. The seeding density of mesenchymal stem cells was 5×10 6 After the acellular matrix hydrogel was formed, an equal volume of liver differentiation medium was added to induce hepatic differentiation of mesenchymal stem cells. After 23 days, micro-liver tissues of the control group were obtained and the expression of liver function-related genes at the (a) mRNA and (b) protein levels was measured. The results are shown in the figure. Figure 6 Group I and Figure 7 Group I.

[0063] (2) Preparation of micro-liver tissue in control group 2 (compared with Example 3, the 3D-printed vascular structure in this control group did not include a hollow tube structure; compared with control group 1, this control group included 3D-printed hexagonally distributed vascular endothelial cells)

[0064] The 3D printed hexagonally distributed vascular endothelial cells without hollow tube structures were mixed with the 50 μm diameter mesenchymal stem cell spheres prepared in Example 1 at a cell density of 2:1, embedded in the liver decellularized matrix hydrogel, and incubated at 37 degrees Celsius for 1 hour. The seeding density of the mesenchymal stem cells was 5×10 6 After the acellular matrix hydrogel was formed, an equal volume of liver differentiation medium was added to induce hepatic differentiation of mesenchymal stem cells. After 23 days, two micro-liver tissues were obtained from the control group and the expression of liver function-related genes at the (a) mRNA and (b) protein levels was measured. The results are shown in Figure 2. Figure 6 Group II.

[0065] (4) Preparation of micro-liver tissue in control group 3 (compared with Example 3, the 3D-printed vascular structure in this control group did not contain vascular endothelial cells; compared with control group 1, this control group contained a hollow 3D-printed conduit structure)

[0066] The vascular endothelial cells were mixed with the mesenchymal stem cell spheres with a diameter of 50 microns prepared in Example 1 at a ratio of 2:1, embedded in the liver decellularized matrix hydrogel, and mixed into the gaps of the 3D printed vascular structure without vascular endothelial cells, and incubated at 37 degrees Celsius for 1 hour. Among them, the 3D printed biomimetic liver lobule vascular structure does not contain vascular endothelial cells. After the decellularized matrix hydrogel was gelled, an equal volume of liver differentiation medium was added to begin inducing the liver differentiation of mesenchymal stem cells. After 23 days, 3 micro liver tissues of the control group were obtained, and the expression of liver function-related genes at the (a) mRNA and (b) protein levels was measured. The results are shown in the figure. Figure 7 The results are shown in Group II.

[0067] Depend on Figure 6 It can be seen that compared with the randomly distributed vascular endothelial cells in control group 1, the 3D printed hexagonally distributed vascular endothelial cells without hollow channels in control group 2 can significantly promote the hepatic differentiation of mesenchymal stem cells.

[0068] Depend on Figure 7 It can be seen that the 3D printed vascular structure without vascular endothelial cells in control group 3 can significantly promote the hepatic differentiation of mesenchymal stem cells compared with the control group 1 that does not contain a vascular structure.

[0069] Example 5: Treatment of Acute Liver Failure Induced by Carbon Tetrachloride Using the Micro-Liver Tissue of the Present Invention

[0070] Thirty Balb / c mice (female, 8 weeks old, 18-21 g) were randomly divided into five groups of six mice each. An acute liver failure model was established by intraperitoneal injection of a 35:100 volume ratio of carbon tetrachloride / olive oil mixed solvent (dose: 4 μl / g mouse body weight). 24 hours later, the biomimetic micro-liver tissue prepared in Example 3 was subcutaneously implanted into the inguinal fat pad rich in blood vessels of the acute liver failure mice. The implanted micro-liver tissue contained 1×10 6 After one day, the liver necrosis area was significantly reduced and the oxidative stress and inflammatory storm were alleviated by the mesenchymal stem cells that differentiated into hepatocytes. Figure 8 Middle V group).

[0071] Example 6: Treatment of acute liver failure caused by excessive intake of acetaminophen using the micro-liver tissue of the present invention

[0072] Thirty Balb / c mice (female, 8 weeks old, 18-21 g) were randomly divided into five groups of six mice each. After fasting for 24 hours, the mice were intraperitoneally injected with 10 mg / ml acetaminophen solution (dose: 40 μl / g mouse body weight) to establish a drug-induced acute liver failure model. Twenty-four hours later, the biomimetic micro-liver tissue prepared in Example 3 was subcutaneously implanted into the inguinal fat pad rich in blood vessels of the acute liver failure mice. The implanted micro-liver tissue contained 1x10 6 After 3 days, the liver necrosis and congestion area were significantly reduced, the inflammatory storm was relieved, the liver cells proliferated significantly, the antioxidant capacity was improved, and the liver function was restored. Figure 9 Group IV).

[0073] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a bionic micro-liver tissue, characterized in that: include: Resuspending vascular endothelial cells and stem cell spheres in the same hydrogel to obtain a mixture; The stem cell sphere is a mesenchymal stem cell sphere; stacking the bionic liver lobule-like vascular structures; adding the mixture into the gaps of the bionic liver lobule-like vascular structures, incubating, and adding liver differentiation medium to induce liver differentiation of mesenchymal stem cells after the hydrogel is gelled, thereby obtaining bionic micro-liver tissues; The method for preparing the bionic liver lobule-like vascular structure comprises: Step 1) preparing an outer layer support ink and an inner layer sacrificial ink respectively; Step 2) performing 3D coaxial bioprinting using the outer support ink and the inner sacrificial ink according to a preset hexagonal liver lobule pattern with a circular hole channel in the middle and equally divided radially from the circular hole channel to the surrounding areas, wherein the outer support ink and the inner sacrificial ink form a hexagonal hollow circular hole structure including equally divided liver sinusoidal areas; Step 3) placing the hexagonal hollow circular hole structure in a cross-linking solution to cross-link the outer layer support ink into shape, removing the inner layer sacrificial ink, and obtaining a bionic liver lobule-like vascular structure in which both the central vein and the hepatic sinusoid region are hollow tube structures; the bionic liver lobule-like vascular structure has a hexagonal interconnected vascular network; The diameter of the stem cell sphere is 50-100 μm; the cell density ratio of the vascular endothelial cells and the stem cell sphere is 2:

1.

2. The preparation method according to claim 1, characterized in that: In step 1), the outer supporting ink comprises vascular endothelial cells and the following components by mass fraction: 0.1-2% sodium alginate, 1-8% gelatin and 0.1-10% fibrinogen; the inner sacrificial ink comprises 1-8% gelatin, 0.1-2% sodium hyaluronate and 1-100 units / ml thrombin.

3. The preparation method according to claim 1, characterized in that: In step 2), the printing conditions are: The extrusion pressures of the supporting ink and the sacrificial ink are 0.01-0.1 MPa and 0.1-0.5 MPa respectively, and the temperature is 15-25° C.; The inner layer printing needle has a diameter of 0.1-1 mm, and the outer layer printing needle has a diameter of 0.5-5 mm. The inner layer printing needle has a diameter smaller than the outer layer printing needle. The inner and outer needle temperatures are both 15-20° C., which is lower than the ink temperature.

4. The preparation method according to claim 1, characterized in that: In step 3), the cross-linking solution includes 40 mmol / L calcium chloride, 0.9 wt% sodium chloride and 8 units / ml thrombin; The cross-linking solution is a solution pre-cooled at 4°C, and the cross-linking time is 30 minutes; The method for removing the inner layer sacrificial ink comprises: immersing the cross-linked structure in a phosphate buffer solution preheated at 37°C.

5. The preparation method according to claim 1, characterized in that: The vascular endothelial cells are human umbilical vein endothelial cells, or vascular endothelial cells derived from liver sinusoids, arteries, dermal microvessels, and stem cells differentiated.

6. The preparation method according to claim 1, characterized in that: The hydrogel is a liver decellularized matrix hydrogel, or a hydrogel made of the following raw materials: gelatin, hyaluronic acid, polyethylene glycol and its derivatives.

7. Use of the mini liver tissue prepared by the preparation method according to any one of claims 1 to 6 in the safety assessment of hepatotoxic drugs or cosmetics.

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