CN117844738B - A bionic micro liver tissue and its preparation method and application - Google Patents
A bionic micro liver tissue and its preparation method and application Download PDFInfo
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- CN117844738B CN117844738B CN202410027897.0A CN202410027897A CN117844738B CN 117844738 B CN117844738 B CN 117844738B CN 202410027897 A CN202410027897 A CN 202410027897A CN 117844738 B CN117844738 B CN 117844738B
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Abstract
本发明涉及生物技术领域，尤其涉及一种仿生微肝组织及其制备方法和应用。相比于常规的细胞分化后再共培养的策略，本发明直接对共培养中的间充质干细胞球诱导分化，不仅提高了间充质干细胞的肝向分化效率，也简化了操作步骤。同时，异位皮下植入本发明微肝组织也显示出显著的肝衰竭治疗效果。
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
The invention relates to the technical field of biology, in particular to a bionic micro-liver tissue and a preparation method and application thereof.
Background
The stem cells are often induced to differentiate into hepatocyte-like cells in a two-dimensional culture dish with insufficient maturity, and are difficult to perform normal liver functions, so that the curative effect of treating acute liver failure is limited. The cytosphere formed by multicellular aggregation has higher hepatic differentiation potential, but the size effect is freshly reported, and the invention realizes efficient hepatic cell differentiation by controlling the size of the mesenchymal stem cell sphere. In addition, vascularization is also the key of timely exerting curative effect on artificial liver tissues, and the conventional method directly mixes vascular endothelial cells with differentiated hepatocyte-like cells simply and cannot simulate specific vascular structures in liver lobules well. Based on the method, the invention also utilizes the 3D coaxial printing vascular endothelial cells to construct a liver small She Fangsheng vascular structure, and further improves the liver differentiation efficiency of the mesenchymal stem cells by co-culturing with the mesenchymal stem cell spheres, and the constructed vascularized micro-liver tissue remarkably promotes liver regeneration of the acute liver failure mice after subcutaneous transplantation.
Mesenchymal stem cells are typically differentiated into hepatocyte-like cells in conventional two-dimensional dishes via an induction medium, and in order to increase hepatic differentiation efficiency of mesenchymal stem cells, they are often embedded in three-dimensional hydrogels or engineered into multicellular cytoballs to better mimic the microenvironment of the cells in vivo. In addition, hepatocyte function can also be enhanced by co-culturing hepatocyte-like cells and vascular endothelial cells, wherein the ratio and spatial distribution of vascular endothelial cells are important influencing factors. Liver lobules are the smallest functional unit of liver tissue, with a hexagonal network of communicating vessels. The prior art mainly distributes liver cells and vascular endothelial cells alternately by presetting extrusion type biological printing or digital light treatment so as to simply simulate the hexagonal structure of liver lobules.
Although the cytoball formed by multicellular aggregation has higher hepatic differentiation potential than single-cell cells with the same number, the prior art schemes have little attention on the size effect because the size of the cytoball is highly correlated with the function of the cytoball, so that the curative effect lacks consistency, and most of prior art schemes induce mesenchymal stem cells to undergo hepatic differentiation in advance and then co-culture with vascular endothelial cells, thus the operation is complex. In addition, although bionic liver lobule structures can be constructed by preset extrusion type biological printing and digital light processing, vascular endothelial cells are only distributed alternately with liver cells to form a hexagonal structure, and complete communication and hollow vascular structures are lacked.
Disclosure of Invention
In view of the above, the invention provides a bionic micro-liver tissue and a preparation method and application thereof. The bionic liver lobule constructed by the invention has a hexagonal communicated vascular network, is co-cultured with mesenchymal stem cell spheres and induces hepatic differentiation of the mesenchymal stem cell spheres, and successfully prepares the micro liver tissue with the polar liver failure treatment effect.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of a bionic liver small She Yang vascular structure, which comprises the following steps:
Step 1) respectively preparing outer layer supporting ink and inner layer sacrificial ink;
Step 2) carrying out 3D coaxial biological printing by using the outer layer supporting ink and the inner layer sacrificial ink according to a preset hexagonal pattern with a middle round hole and twelve radial equally divided peripheries, so as to obtain a hexagonal structure;
and 3) placing the hexagonal structure in a crosslinking solution to crosslink and shape the outer layer supporting ink, and removing the inner layer sacrificial ink to obtain the bionic liver small She Yang vascular structure.
In the step 1), the outer layer supporting ink comprises vascular endothelial cells and the following components in percentage by mass of 0.1-2% sodium alginate, 1-8% gelatin and 0.1-10% fibrinogen, and the inner layer sacrificial ink comprises 1-8% gelatin, 0.1-2% sodium hyaluronate and 1-100 units/ml thrombin.
In some embodiments, the outer layer supporting ink comprises vascular endothelial cells, 1.5% sodium alginate, 5% gelatin, and 0.1% fibrinogen, and the inner layer sacrificial ink comprises 7.5% gelatin, 1.5% sodium hyaluronate, and 8 units/ml thrombin.
In the invention, in the step 2), the printing conditions are as follows:
The extrusion air pressure of the supporting ink and the sacrificial ink is respectively 0.01-0.1 MPa, 0.1-0.5 MPa, the temperature is 15-25 ℃, the diameter of the needle head printed on the inner layer is 0.1-1 mm, the diameter of the needle head printed on the outer layer is 0.5-5 mm, and the temperature of the needle head is 15-20 ℃. Wherein, the diameter of inlayer printing syringe needle is less than the diameter of skin printing syringe needle, and inside and outside syringe needle temperature is less than the temperature of ink.
In some embodiments, the printing conditions are:
the extrusion air pressure of the supporting ink and the sacrificial ink is 0.05MPa, 0.35MPa and the temperature is 25 ℃, the diameter of the needle head printed on the inner layer is 0.26mm, the diameter of the needle head printed on the outer layer is 0.8mm, and the temperature of the needle head is 20 ℃.
In the step 3), the crosslinking solution comprises 40mmol/L calcium chloride, 0.9wt% sodium chloride and 8 units/mL thrombin, wherein the crosslinking solution is a solution precooled at 4 ℃ and the crosslinking time is 30min;
The method for removing the inner layer sacrificial ink comprises the step of immersing the crosslinked structure in a preheated phosphoric acid buffer solution at 37 ℃.
The invention also provides a bionic hepatic lobular vein tube structure prepared by the preparation method.
The bionic hepatic lobular vein structure printed by the invention is a hepatic lobular hexagonal structure of a hollow circular hole pipeline, and the structure comprises equally divided hepatic sinus regions, so that the number of divided hepatic lobular vein structures can be determined according to actual conditions by a person skilled in the art. In the invention, the equal division may be 6-96 equal divisions, and specifically may be 6, 12, 24, 48 or 96 equal divisions.
The invention also provides a preparation method of the bionic micro-liver tissue, which comprises the following steps:
the vascular endothelial cells and the stem cell pellets are resuspended in the same hydrogel to obtain a mixture;
Adding the mixture into a gap of a vascular structure of a bionic liver small She Yang, incubating, and adding a liver differentiation medium after the hydrogel is gelled to induce hepatic differentiation of mesenchymal stem cells to obtain a bionic micro-liver tissue;
the stem cell pellet comprises one or more of mesenchymal stem cell pellet, embryonic stem cell pellet, induced pluripotent stem cell pellet and hepatoblast cell pellet.
In some embodiments of the invention, the stem cell pellet is a mesenchymal stem cell pellet. Specifically, the mesenchymal stem cell spheres are obtained through micropore culture, and can also be prepared through other methods, such as low-adsorption culture dish culture, culture on a STEMCELL Technologies Inc. Aggresell TM product, porous culture dish culture, hanging drop culture, microsphere culture prepared by a microfluidics technology, dynamic culture of a stirred bioreactor and the like.
The invention obtains mesenchymal stem cell spheres with different sizes by controlling the cell inoculation amount and using micropores with different specifications. In some embodiments, the stem cell sphere has a diameter of 50-150 μm, specifically 50 μm, 100 μm, 150 μm. The invention compares the influence of the sizes (50 mu m, 100 mu m, 150 mu m and 200 mu m) of each cell sphere within the oxygen uptake limit distance (200 mu m) on hepatic differentiation, determines the diameter size of 50 mu m as the optimal size, and can maximize the hepatic differentiation efficiency of the mesenchymal stem cell sphere.
In the invention, the cell number ratio of the vascular endothelial cells to the stem cell pellets is (0-4): 1, and in some embodiments, the ratio is specifically 2:1, 3:1 or 4:1. In one specific embodiment, vascular endothelial cells and mesenchymal stem cell spheres (with the diameter of 50 microns) are co-cultured in a hepatic decellularized matrix hydrogel in different proportions (0:1, 1:1, 2:1 and 4:1) and hepatic differentiation of the mesenchymal stem cells is induced, and the expression difference of liver function related genes on the level of (a) mRNA and (b) protein is compared, so that the co-culture proportion of the vascular endothelial cells and the mesenchymal stem cell spheres 2:1 is shown to obviously promote hepatic differentiation of the mesenchymal stem cells.
In the invention, the bionic liver small She Yang vascular structure is a liver small She Yang vascular structure prepared by the preparation method provided by the invention, and has a hexagonal hollow communicated vascular structure.
In the present invention, the vascular endothelial cells include endothelial cells derived from umbilical vein, hepatic sinus, artery, dermal layer microvasculature or vascular endothelial cells differentiated from stem cells. In some embodiments, the vascular endothelial cells are human umbilical vein endothelial cells.
In the present invention, the incubation time was 37 ℃ and the induction time of hepatic differentiation was 23 days.
In the invention, the hydrogel is a hepatic decellularized matrix hydrogel or a hydrogel prepared from gelatin, hyaluronic acid, polyethylene glycol and derivatives thereof. In some embodiments, the hydrogel is a liver decellularized matrix hydrogel prepared by repeatedly freeze thawing 1 cubic centimeter pig liver pieces 3 times at-80 ℃ and room temperature, while washing out blood cells with ultrapure water. Subsequently, stirring was continued in a decellularized complex solution of 1% Triton x-100/0.1% ammonia for 3 days with water change 3 times per day. Finally, the mixture was freeze-dried and ground to a powder, then weighed, and digested in a weight ratio of 10:1 liver extracellular matrix to pepsin in 0.1M sodium hydroxide solution for 36 hours. The liver decellularized matrix hydrogel was used at a concentration of 10 mg/ml and a pH of 7.2.
The invention also provides the bionic micro-liver tissue prepared by the preparation method.
The bionic micro-liver tissue prepared by the invention has the diameter of 100 micrometers-1 cm and the height of 100 micrometers-1 cm, and can contain 6, 12, 24, 48 or 96 equally divided liver sinus regions.
The invention also provides a method for treating acute liver failure, which comprises the step of implanting the micro-liver tissue. The implantation site includes the subcutaneous blood vessel-rich fat pad of groin, mesentery, liver capsule, lymph node, etc. The acute liver failure includes carbon tetrachloride, acetaminophen, D-galactosamine/lipopolysaccharide induced liver failure or liver failure caused by liver ischemia reperfusion.
In the present invention, the micro-liver tissue to be implanted is of modular design, and the number may be one or more stacks, wherein the number of hepatocyte-like cells contained in each micro-liver tissue is 1×10 6-1×109.
The invention also provides application of the bionic micro-liver tissue in evaluating the safety of hepatotoxic drugs or cosmetics. The liver toxicity medicine comprises a traditional Chinese medicine formula, a Chinese patent medicine or other western medicines with liver toxicity substances.
The 3D coaxial bioprinting vascular endothelial cells construct a hexagonal communication vascular network of bionic liver lobules, and co-culture the vascular network and mesenchymal stem cell spheres, and the hepatic differentiation of the mesenchymal stem cells is directly induced in the co-culture process, so that the micro liver tissue with the polar liver failure treatment effect is successfully prepared. Not only improves the hepatic differentiation efficiency of the mesenchymal stem cells, but also simplifies the operation steps. Experimental results show that the micro liver tissue constructed by the invention successfully promotes the repair and regeneration of liver tissue in mice with acute liver failure induced by carbon tetrachloride and acetaminophen.
Drawings
FIG. 1 shows a flow chart for the construction of micro-liver tissue;
FIG. 2 shows three sizes of mesenchymal stem cell spheres prepared from single cells and micropores, S cell spheres (diameter: 50 microns), M cell spheres (diameter: 100 microns) and L cell spheres (diameter: 150 microns), a is cell activity (scale: 100 microns), b-f is a representation of hepatic differentiation, wherein b-c is liver function-related gene mRNA expression level, and e-f is protein level (scale: 50 microns);
FIG. 3 shows (a) morphology (large scale: 200. Mu.m; small scale: 50. Mu.m) and (b-c) hepatic differentiation (hepatic function-related genes at mRNA and protein levels) (scale: 100. Mu.m) of three sizes of mesenchymal stem cell spheres (XS cell spheres (diameter: 25. Mu.m), S cell spheres (diameter: 50. Mu.m) and XL cell spheres (diameter: 200. Mu.m)) prepared by microwells;
FIG. 4 shows a 3D coaxial printing bionic liver lobule-like vascular system and a temperature-sensitive liver decellularized matrix hydrogel, (b) a scanning electron microscope shows a hollow tubular structure, a ruler of 200 microns, (c) cell death staining shows that 3D printed vascular endothelial cells have better cell activity, the bionic liver lobules can maintain stable structure in a culture medium, the ruler of 200 microns, (D) vascular endothelial cells singly embedded in the liver decellularized matrix hydrogel under a bright field microscope, vascular endothelial cells and mesenchymal stem cell spheres co-cultured in the stem decellularized matrix hydrogel, 3D printed vascular endothelial cells and mesenchymal stem cell spheres filled in gaps between liver and liver sinuses, and the ruler of 500 microns;
FIG. 5 shows the difference in expression of genes related to liver function at the (a) mRNA and (b) protein levels on a scale of 50. Mu.m, by co-culturing vascular endothelial cells with 50 μm diameter mesenchymal stem cell spheres in different ratios (0:1, 1:1, 2:1 and 4:1) in hepatic decellularized matrix hydrogel and inducing hepatic differentiation of the mesenchymal stem cells;
FIG. 6 shows the expression difference of genes related to liver function on the level of (a) mRNA and (b) protein, scale of 100 microns, by co-culturing vascular endothelial cells randomly distributed or hexagonally distributed in comparative examples 1-2 and mesenchymal stem cell spheres in a 2:1 ratio in a matrix hydrogel containing liver decellularization and inducing liver differentiation of the mesenchymal stem cells;
FIG. 7 shows that comparative examples 3-4 vascular endothelial cells and mesenchymal stem cell spheres were randomly mixed into a hepatic decellularized matrix hydrogel with or without a hexagonal hollow tube at a ratio of 2:1 for co-culture, and hepatic differentiation of mesenchymal stem cells was induced, and the expression difference of liver function-related genes on the (a) mRNA and (b) protein levels was measured at 100 μm scale;
FIG. 8 shows (a-b) liver tissue morphology and abnormal area statistics of different treatment groups, scale: 100. Mu.m, (c) liver tissue oxidative stress level of different treatment groups, (d) expression difference of inflammatory factor genes (Nos 2 and Tnfa) on mRNA level in liver tissues of different treatment groups, (e) expression level of cyclooxygenase-2 in liver tissues of different treatment groups by immunofluorescent staining, scale: 200. Mu.m, (f) content of tumor necrosis factor TNF-. Alpha., inducible nitric oxide synthase iNOS and anti-inflammatory factor (interleukin IL-10) in liver tissues of different treatment groups by ELISA;
FIG. 9 (a) forms of liver tissues of different treatment groups, (b) differences in expression of inflammatory factor genes (Tnfa, il1b, nos. 2 and Il 6), antioxidant genes (Sod 1 and Sod 2) and liver function-related genes (Alb and Cyp3a 11) on mRNA in liver tissues of different treatment groups, (c) immunoblots for detecting the amounts of TNF- α and iNOS relative to GAPDH, respectively, (d) ELISA for detecting the amounts of TNF- α in liver tissues of different treatment groups, and (e) immunofluorescent staining for detecting the expression levels of granulocyte marker (lymphocyte antigen-6G) and cell proliferation marker Ki-67, respectively, in liver tissues of different treatment groups, on a scale of 200. Mu.m.
Detailed Description
The invention provides a bionic micro-liver tissue and a preparation method and application thereof. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
The invention is further illustrated by the following examples:
EXAMPLE 1 preparation of mesenchymal Stem cell spheres
Mesenchymal stem cells (10 k, 25k and 50 k) with different inoculum sizes were inoculated into an array of microwells (length x width x height: 0.5 mm x 0.22 mm, total of 100 microwells), and after centrifugation at 600rcf for 5 minutes, the mesenchymal stem cell pellets (FIG. 2 a-b) with diameters of 50 microns (S cell pellet), 100 microns (M cell pellet) and 150 microns (L cell pellet) were prepared in sequence. Wherein 10k represents seeding 10000 cells/microwell array.
The results of detecting the liver function-related gene mRNA and protein expression levels of single cells and the above mesenchymal stem cell pellet show that compared with single cells (single cells) cultured in a two-dimensional culture dish, the mesenchymal stem cell pellet has better hepatic differentiation potential (expression of liver-related gene at transcription level and translation level), and the differentiation efficiency of 50-micron diameter cell pellet (S cell pellet) is higher (fig. 2 c-f).
Meanwhile, the same number of cells (10 k) were inoculated into microwell arrays of different sizes (length×width×height: 0.33 mm×0.3 mm, total of 225 microwells; length×width×height: 0.5 mm×0.5 mm×0.22 mm, total of 100 microwells; length×width×height: 1.3 mm×1.3 mm×0.9 mm, total of 10 microwells), and after centrifugation for 5 minutes, mesenchymal stem cell spheres (FIG. 3 a) of 25 μm (XS cell sphere), 50 μm (S cell sphere) and 200 μm (XL cell sphere) in this order were obtained by overnight culture. The results of the liver-associated gene mRNA and protein expression level detection showed that 50 micron diameter cell spheres (S-cell spheres) were the most capable of hepatic differentiation (FIGS. 3 b-c).
Example 23D coaxial bioprinting bionic hepatic lobular vein tube structure
1) The outer layer supporting ink (ESI) is composed of 1.5% sodium alginate, 5% gelatin, 0.1% fibrinogen and vascular endothelial cells, and the inner layer sacrificial ink (ISI) is mainly composed of 7.5% gelatin, 1.5% sodium hyaluronate and 8 units/ml thrombin.
2) The diameters of the inner layer needle head and the outer layer needle head are respectively 0.26 millimeter and 0.8 millimeter, the temperature is kept at 20 ℃, the inner layer sacrificial ink and the outer layer sacrificial ink are respectively extruded to a receiving plate with temperature control of 4 ℃ through air pressure of 0.05 MPa and 0.35 MPa at 25 ℃, and a twelve-equal-part hexagonal hollow round hole structure is printed.
3) The printed structure was immersed in a pre-chilled cross-linking solution (consisting of 40mmol/L calcium chloride, 0.9% sodium chloride and 8 units/ml thrombin) at 4 degrees celsius for 30 minutes to gel the outer support ink. Finally, the molded structure was immersed in a pre-heated 1 x phosphate buffer solution at 37 ℃ to remove the sacrificial ink of the inner layer, thereby forming an empty tube structure, and a bionic hepatic lobular vein structure was obtained (see fig. 4).
EXAMPLE 3 preparation of bionic micro-liver tissue according to the invention
Vascular endothelial cells and 50 μm diameter mesenchymal stem cell spheres prepared in example 1 were embedded in the hepatic decellularized matrix hydrogel at different cell density mixing ratios (0:1, 1:1, 2:1 and 4:1), respectively, the seeding density of the mesenchymal stem cells was 5×10 6 cells/ml, and mixed into the 3D printed biomimetic hepatic lobular vein structural gap of example 2, and incubated at 37 degrees celsius for 1 hour. After the acellular matrix hydrogel is gelled, adding an equal volume of liver differentiation medium, and starting to induce hepatic differentiation of the mesenchymal stem cells, and culturing for 23 days (i.e. a co-culture process) to obtain the micro-liver tissue (see figure 5). Wherein, the density of the vascular endothelial cells in the 3D printing bionic liver lobular vein structure is consistent with the density of the vascular endothelial cells mixed in the acellular matrix hydrogel.
EXAMPLE 4 preparation of micro liver tissue by Co-culturing vascular endothelial cells and mesenchymal Stem cells of different morphologies
(1) Preparation of control group 1 micro-liver tissue (compared to example 3, the control group does not contain 3D printed vascular structures)
The random distribution vascular endothelial cells were mixed with the 50 micron diameter mesenchymal stem cell spheres prepared in example 1 at a ratio of 2:1, embedded in the hepatic decellularized matrix hydrogel, and incubated at 37 degrees celsius for 1 hour. Wherein, the inoculation density of the mesenchymal stem cells is 5×10 6 cells/ml. After gel formation of the decellularized matrix hydrogel, adding an equal volume of liver differentiation medium, starting to induce hepatic differentiation of mesenchymal stem cells, obtaining a micro liver tissue of a control group 1 after 23 days, and measuring the expression condition of liver function related genes at the level of (a) mRNA and (b) protein, wherein the results are shown in a group I in FIG. 6 and a group I in FIG. 7.
(2) Preparation of control group 2 micro liver tissue (3D printed vascular structure does not contain hollow conduit structure in comparison with example 3; control group contains vascular endothelial cells in a hexagonal distribution in 3D printed compared with control group 1)
Vascular endothelial cells without hollow pipeline structures distributed in a 3D printing mode and 50-micrometer-diameter mesenchymal stem cell spheres prepared in example 1 are mixed in a ratio of cell density of 2:1, embedded in hepatic decellularized matrix hydrogel and incubated for 1 hour at 37 ℃. Wherein, the inoculation density of the mesenchymal stem cells is 5×10 6 cells/ml. After gel formation of the decellularized matrix hydrogel, adding an equal volume of liver differentiation medium, starting to induce hepatic differentiation of mesenchymal stem cells, obtaining micro liver tissue of a control group 2 after 23 days, and measuring the expression condition of liver function related genes at the levels of (a) mRNA and (b) protein, wherein the result is shown in a group II in fig. 6.
(4) Preparation of control 3 micro liver tissue (the 3D printed vascular structure in the control does not contain vascular endothelial cells compared to example 3; the control contains a hollow 3D printed tubing structure compared to control 1)
Vascular endothelial cells were mixed with 50 micron diameter mesenchymal stem cell spheres prepared in example 1 at a ratio of 2:1, embedded in hepatic decellularized matrix hydrogel, and mixed into the gap of 3D printed vascular structures without vascular endothelial cells, and incubated at 37 degrees celsius for 1 hour. Wherein, the 3D printing bionic liver lobular vein structure does not contain vascular endothelial cells. After gel formation of the decellularized matrix hydrogel, adding an equal volume of liver differentiation medium, starting to induce hepatic score of mesenchymal stem cells, obtaining 3 micro liver tissues of a control group after 23 days, and measuring the expression condition of liver function related genes at the level of (a) mRNA and (b) protein, wherein the result is shown as a result of a group II in FIG. 7.
As can be seen from fig. 6, compared with the vascular endothelial cells randomly distributed in the control group 1, the vascular endothelial cells without hollow ducts, which are distributed in the control group 2 in a 3D printing manner, can significantly promote hepatic differentiation of mesenchymal stem cells.
As can be seen from fig. 7, the 3D printed vascular structure of the control group 3 without vascular endothelial cells significantly promoted hepatic differentiation of mesenchymal stem cells compared to the scheme of the control group 1 without vascular structure.
EXAMPLE 5 treatment of carbon tetrachloride-induced acute liver failure Using the micro-liver tissue of the present invention
30 Balb/c mice (female, 8 weeks old, 18-21 g) were randomly divided into 5 groups of 6. The acute liver failure model was constructed by intraperitoneal injection of 35:100 volume ratio of carbon tetrachloride/olive oil mixed solvent (dose: 4 μl/g mouse body weight). After 24 hours, the bionic micro-liver tissue prepared in example 3 was subcutaneously implanted in the inguinal blood vessel-rich fat pad of acute liver failure mice, wherein the implanted micro-liver tissue contained 1×10 6 hepatic differentiated mesenchymal stem cells, and a significant reduction in liver necrosis area and alleviation of oxidative stress and inflammatory storm could be observed after 1 day (group V in fig. 8).
EXAMPLE 6 treatment of acute liver failure due to excessive acetaminophen uptake Using micro liver tissue of the invention
30 Balb/c mice (female, 8 weeks old, 18-21 g) were randomly divided into 5 groups of 6. The mice were fasted for 24 hours and then were intraperitoneally injected with 10 mg/ml acetaminophen solution (dose: 40 μl/g mouse body weight) to construct a drug-induced acute liver failure model. After 24 hours, the bionic micro-liver tissue prepared in example 3 was subcutaneously implanted in the inguinal blood vessel-rich fat pad of acute liver failure mice, wherein the implanted micro-liver tissue contained 1×10 6 hepatic differentiated mesenchymal stem cells, and after 3 days, significant reduction of liver necrosis and blood stasis area, alleviation of inflammatory storm, significant proliferation of liver cells, improvement of antioxidant capacity and recovery of liver function were observed (group IV in fig. 9).
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (7)
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