A method of inducing hepatic oval cell lines to form functional hepatoid-like organoid tissue on a liver acellular bio-scaffold

Abstract

The present application relates to a method for inducing liver oval cell line to form functional liver-like organ tissue on a liver acellular biological scaffold, belonging to the field of biotechnology, wherein the liver oval cell line (HOCL) is used as seed stem cells, and is implanted into a regenerative liver acellular biological scaffold (R-DLS) with biological activity; according to the in-vitro simulation of the development of a body-like liver organ microenvironment concept, a conditional culture solution capable of inducing HOCL liver function differentiation and reconstructing a functional liver organ structure is added, and a functional liver-like organ tissue is constructed in an in-vitro 3D culture system. In the method, the HOCL in the R-DLS shows its differentiation into functional hepatocytes in 2-24h in the in-vitro 3D culture system, and the liver-like organ tissue can be formed in 21 days. The "new liver" constructed by the method has important clinical conversion value for being used as a liver transplantation donor and treating ESLD.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a method for inducing hepatic oval cell lines to form functional liver-like organoid tissues on a liver acellular biological scaffold. BACKGROUND

[0002] End-stage liver disease (ESLD) includes acute liver failure (ALF) and chronic liver cirrhosis, which has a high mortality rate and seriously endangers human health. It is also a major disease in China. The only effective treatment at present is liver transplantation, but liver transplantation is difficult to be widely used due to the shortage of donors. Therefore, finding an effective liver function replacement technology is the key to solving this problem. At present, with the help of in vitro temporary assistance or replacement of liver and other artificial livers, such as bioartificial liver (BAL), although various harmful substances generated or increased due to liver failure can be removed, etc., only the symptoms of late-stage patients can be temporarily improved, and the full replacement of liver function cannot be achieved. In recent years, there have been more studies on engineering organoid tissues using seed cells and normal organ scaffolds, but there are still relatively few successful construction of liver organs with biological activity in vitro. The main difficulties in constructing organ tissues in vitro using organ scaffolds and seed cells are: (1) optimization of scaffold materials; (2) selection of seed cells; (3) establishment of in vitro 3D culture microenvironment.

[0003] The scaffold for liver tissue reconstruction mainly includes biological scaffold and physical scaffold. Among them, the physical scaffold is made into the shape of the liver scaffold by using materials with good biocompatibility, but the existing 3D printing technology cannot construct precise liver complex pipeline (such as portal vein, hepatic artery, bile duct) system. The biological scaffold has become the mainstream direction of current research because it uses natural liver scaffolds to completely retain the pipeline system of the liver, but due to the need to ensure physiological activity during the decellularization process of the biological scaffold, there are still relatively few reports on clinically implementable methods and achievements for successfully constructing liver tissues using biological scaffolds.

[0004] Seed cells for liver construction need to have the potential to differentiate into hepatocytes and cholangiocytes. At present, the academic circle uses adult hepatocytes and mesenchymal stem cells (MSCs), among which, due to the poor survival condition of in vitro long-term culture of hepatocytes in the existing methods, and the lack of potential of MSCs to differentiate into a large number of functional hepatocytes, the application of these cells is greatly limited.

[0005] The establishment of in vitro microenvironment mainly lies in the establishment of the condition medium simulating the growth environment of liver and the culture condition, wherein the research of the culture medium is currently mainly focused on the culture of hepatocytes, and the culture medium for constructing liver tissue is rarely reported.

[0006] CN104894066A discloses a method for reconstructing artificial liver in vitro by using stem cell group NG2+HSC as seed cells, but the method uses liver-derived stem / progenitor cells (NG2+HSC) expressing neural-glial antigen 2 as seed cells, the preparation method of the seed cells is complex, is not conducive to large-scale use, and in the process of reconstructing artificial liver in vitro by (NG2+HSC), three different culture media need to be replaced, the operation process is complicated and difficult to control, the reconstruction of artificial liver takes a long time, and is not conducive to large-scale application.

[0007] The hepatocyte oval cell line (HOCL) is a liver stem cell with multi-differentiation potential, which can be activated to differentiate into hepatocytes and bile duct cells and even other tissue cells under certain conditions, and the hepatocyte oval cell can be obtained through a commercial channel, which is not only convenient in source, but also can be quickly (about 1-24h) differentiated into a large number of mature hepatocytes, bile duct cells and functional liver tissue structures, and the strong functional differentiation potential lays a theoretical foundation for the construction of functional liver-like tissue in vitro, therefore, using the hepatocyte oval cell line as seed cells and constructing liver tissue on a biological scaffold through a specific condition medium can solve the problem of bionic liver supply, and the current problems to be solved are the condition medium that can be realized in clinic and the cost that can be accepted and the in vitro tissue culture method. SUMMARY

[0008] Therefore, the purpose of the present application is to provide a method for inducing hepatocyte oval cell line to form functional liver-like organ tissue on a liver acellular biological scaffold.

[0009] In order to achieve the above purpose, the present application provides the following technical scheme:

[0010] 1. A method for inducing hepatocyte oval cell line to form functional liver-like organ tissue on a liver acellular biological scaffold, the method comprising the following steps:

[0011] (1) implanting mesenchymal stem cells into the liver acellular biological scaffold through the portal vein, the hepatic artery and the inferior vena cava respectively, adding an induced endothelial cell differentiation culture solution, and statically culturing for 5-7 days, and gradually replacing the induced endothelial cell differentiation culture solution with an induced bile duct cell differentiation culture solution in the later culture period;

[0012] (2) implanting the hepatic oval cell line into the liver acellular biological scaffold treated in step (1) through the bile duct, first static culture for 3-5 days, then dynamic culture for 4-5 days, and gradually replacing the induced bile duct cell differentiation culture solution with induced mature liver cell differentiation culture solution during the dynamic culture;

[0013] (3) implanting the hepatic oval cell line into the liver acellular biological scaffold treated in step (2) through the portal vein, hepatic artery and inferior vena cava, alternating dynamic and static culture for at least 7 days.

[0014] Preferably, the preparation method of the induced endothelial cell differentiation culture solution, induced bile duct cell differentiation culture solution and induced mature liver cell differentiation culture solution is as follows:

[0015] A. adding the cell-free mammalian liver homogenate filtrate of the embryonic or neonatal period into DMEM / F12 culture solution at a volume ratio of 1:4-32, then adding penicillin-G, streptomycin, L-glutamine, non-essential amino acids, sodium pyruvate and HEPES, mixing well, then adding bovine insulin, human transferrin, levothyroxine, triiodothyronine, sodium selenite, putrescine, progestogen and albumin, so that the final concentration of penicillin-G is 100 U / mL, the final concentration of streptomycin is 100 μg / mL, the final concentration of L-glutamine is 5 mM, 1× non-essential amino acids, 1× sodium pyruvate, the final concentration of HEPES is 25 mM, the final concentration of bovine insulin is 3.33 μg / mL, the final concentration of human transferrin is 3.33 μg / mL, the final concentration of levothyroxine is 0.26 μg / mL, the final concentration of triiodothyronine is 0.22 μg / mL, the final concentration of sodium selenite is 3.33 μg / mL, the final concentration of putrescine is 1.06 μg / mL, the final concentration of progestogen is 0.04 μg / mL, and the final concentration of albumin is 0.04 μg / mL, to obtain a basic culture solution;

[0016] B. adding an endothelial cell growth factor to the basic culture solution obtained in step A to obtain an induced endothelial cell differentiation culture solution, with a final concentration of 20 ng / mL;

[0017] C. adding recombinant lgr5, recombinant HNF6 and anti-CEBP-β antibody to the basic culture solution obtained in step A to obtain an induced bile duct cell differentiation culture solution, with a final concentration of 200 ng / mL of recombinant lgr5, 200 ng / mL of recombinant HNF6 and 50 ng / mL of anti-CEBP-β antibody;

[0018] D. To the basal medium obtained in step A, recombinant HNF3β, recombinant HNF4α, anti-CEBP-α antibody, recombinant hepatocyte growth factor, recombinant beta-fibroblast growth factor, octreotide and dexamethasone are added to a final concentration of 200 ng / mL of recombinant HNF3β, 200 ng / mL of recombinant HNF4α, 50 ng / mL of anti-CEBP-α antibody, 100 ng / mL of recombinant hepatocyte growth factor, 50 ng / mL of recombinant beta-fibroblast growth factor, 20 ng / mL of octreotide and 0.1 μM of dexamethasone, to obtain the induction medium for mature hepatocyte differentiation.

[0019] Preferably, in step A, the cell component-removed mammalian embryonic or neonatal liver homogenate filtrate is prepared as follows: the mammalian embryonic or neonatal liver tissue is homogenized, filtered, and the filtrate is collected. The filtrate is then repeatedly frozen and thawed for at least 3 times, each time for at least 30 min, and finally subjected to solid-liquid separation, and the liquid is collected to obtain the cell component-removed mammalian embryonic or neonatal liver homogenate filtrate.

[0020] Preferably,

[0021] In step (1), static culture is carried out at 37°C under 5% CO2 for 5-7 days, and the induction medium for endothelial cell differentiation is added once a day for 3-5 days, 5-10 mL each time, in a culture vessel with a volume of 300-500 mL. Half of the induction medium for endothelial cell differentiation is replaced with the induction medium for bile duct cell differentiation on the 3rd-5th day, and the induction medium for bile duct cell differentiation is used completely on the 4th-6th day.

[0022] In step (2), static culture is carried out at 37°C under 5% CO2 for 3-5 days, and dynamic culture is carried out for 4-5 days. During the static culture, the induction medium for bile duct cell differentiation is added once a day for 3-5 days, 5-10 mL each time, in a culture vessel with a volume of 300-500 mL. Half of the induction medium for bile duct cell differentiation is replaced with the induction medium for mature hepatocyte differentiation on the 1st day of dynamic culture, and the replacement amount of the induction medium for mature hepatocyte differentiation is gradually increased in the following days, and the induction medium for mature hepatocyte differentiation is used completely on the last day.

[0023] In step (3), dynamic and static culture is carried out at 37°C under 5% CO2, and the induction medium for mature hepatocyte differentiation is added once a day for 3-5 days, 5-10 mL each time, in a culture vessel with a volume of 300-500 mL.

[0024] Preferably, the dynamic culture in the dynamic-static alternating culture is specifically as follows: from 8:00 am to 22:00 pm every day, the induced mature hepatocyte differentiation culture solution is input into the liver acellular biological scaffold through a circulating pump, and the liver acellular biological scaffold is rotated at a rotation speed of less than 20 rpm, and the speed of the circulating pump is 20 rpm; the static culture in the dynamic-static alternating culture is specifically as follows: from 22:00 pm to 8:00 am every day, the liver acellular biological scaffold is statically cultured.

[0025] Preferably, in step (1), the mesenchymal stem cells are implanted at a cell amount of not less than 0.3-1.5×10 6 Preferably, in step (2), the liver oval cell line is implanted at a cell amount of not less than 1-5×10 6 Preferably, in step (3), the liver oval cell line is implanted at a cell amount of not less than 0.3-1.5×10 7

[0026] Preferably, the liver acellular biological scaffold is an ex vivo regenerative liver acellular biological scaffold with biological activity.

[0027] Preferably, the ex vivo regenerative liver is an ex vivo regenerative liver of a mammal.

[0028] Preferably, the ex vivo regenerative liver acellular biological scaffold with biological activity is prepared by the following method:

[0029] (1) A donor of an ex vivo regenerative liver is taken, and red blood cells in the ex vivo regenerative liver are washed out by perfusion with physiological saline;

[0030] (2) Cell components in the liver treated in step (1) are dissolved and washed out by perfusion with sterile double-distilled water;

[0031] (3) Cell components in the liver treated in step (2) are washed out by perfusion with a mixture of sodium dodecyl sulfate and a trace digestive solution; the trace digestive solution is a mixture of trypsin and ethylenediaminetetraacetic acid;

[0032] (4) The mixture of sodium dodecyl sulfate and the trace digestive solution in the liver treated in step (3) is washed out by perfusion with sterile double-distilled water;

[0033] (5) The sterile double-distilled water in the liver treated in step (4) is washed out by perfusion with a phosphate buffered saline solution, so as to restore the physiological state, and an ex vivo regenerative liver acellular biological scaffold with biological activity is prepared.

[0034] Preferably, the ex vivo regenerative liver acellular biological scaffold with biological activity is prepared by the following method:

[0035] ​(1) taking the ex situ regenerative liver donor, perfusing the ex situ regenerative liver with 0.9% physiological saline at a speed of 5-200 mL / min for 15-30 min to wash out the red blood cells in the ex situ regenerative liver;

[0036] (2) perfusing the liver treated in step (1) with sterile double distilled water at a speed of 5-200 mL / min for 1-2 h to wash out the cell components in the ex situ regenerative liver;

[0037] (3) continuing to perfuse the liver treated in step (2) with a mixture of 0.5-1% sodium dodecyl sulfate and a trace amount of digestive solution at a speed of 5-200 mL / min for 24-72 h to wash out the cell components in the ex situ regenerative liver, wherein the trace amount of digestive solution is a mixture of 0.0025-0.005% trypsin and 0.001-0.002% ethylenediaminetetraacetic acid;

[0038] (4) continuing to perfuse the liver treated in step (3) with sterile double distilled water at a speed of 5-150 mL / min for 1-2 h to wash out the mixture of sodium dodecyl sulfate and the trace amount of digestive solution in the ex situ regenerative liver;

[0039] (5) continuing to perfuse the liver treated in step (4) with 0.1-0.2 M phosphate buffer at a speed of 5-150 mL / min for 1.5-2 h to wash out the sterile double distilled water and restore the physiological state, thereby obtaining a bioactive ex situ regenerative liver acellular biological scaffold.

[0040] The method provided by the application can overcome the disadvantage that artificial materials cannot construct complex life organs such as livers in the prior art 3D printing technology, and has the advantages of convenient source, simple operation, low cost, short induction and culture time, and the like.

[0041] Additional advantages, objects, and features of the application will be apparent to those skilled in the art upon examination of the following detailed description, it being understood that each BRIEF DESCRIPTION OF DRAWINGS

[0042] In order to make the objectives, technical solutions and advantages of the present application clearer, the preferred embodiments of the present application will be described in detail below with reference to the accompanying drawings, in which:

[0043] Figure 1 Figure 1 is a diagram of the perfusion process for preparing a decellularized bio- scaffold of a regenerative liver organ in Example 1, and a diagram of the pipeline system of the decellularized bio-scaffold of the regenerative liver organ; Figure 1 Figure 1A is a diagram of the perfusion process for preparing a decellularized bio- scaffold of a regenerative liver organ in Example 1; Figure 1 Figure 1B is a diagram of the pipeline system of the decellularized bio-scaffold of the regenerative liver organ)

[0044] Figure 2 Figure 2 is a diagram of the decellularized bio-scaffold of a regenerative liver organ used in Example 2, and diagrams of the liver-like tissue formed at different time periods during culture; Figure 2 Figure 2A is a diagram of the decellularized bio-scaffold of a regenerative liver organ prepared in Example 1; Figure 2 Figure 2B is a diagram of the liver-like tissue formed after a total culture time of 7 days; Figure 2 Figure 2C is a diagram of the liver-like tissue formed after a total culture time of 21 days)

[0045] Figure 3 Figure 3 is a diagram of the ability of the Ov6-stained hepatic oval cell line to be induced to differentiate into CK19-positive cholangiocytes and positive mature hepatocytes in the 3D culture system of the present application in Example 2; Figure 3 Figure 3A is a diagram of the ability of the Ov6-stained hepatic oval cell line to be induced to differentiate into CK19-positive cholangiocytes during static culture in step 2) in Example 2, detected by immunofluorescence double staining; Figure 3 Figure 3B is a diagram of the ability of the Ov6-stained hepatic oval cell line to be induced to differentiate into positive mature hepatocytes (ALB) during dynamic-static alternating culture in step 3) in Example 2, detected by immunofluorescence double staining)

[0046] Figure 4 Figure 4 is a diagram of the secretion of liver function proteins in the supernatant of the functional liver-like tissue constructed in vitro in Example 2, and the test results of transplantation treatment of acute liver failureFigure 4 Figure 1 shows the results of detection of CK19-positive bile duct cell proteins in the supernatant of each functional liver tissue culture; Figure 4 Figure 2 shows the results of detection of mature hepatocyte proteins in the supernatant of each functional liver tissue culture; Figure 4 Figure 3 shows the results of detection of total bilirubin and total bile acid in the supernatant of each functional liver tissue culture; Figure 4 Figure 4 shows the results of testing of the functional liver tissue obtained by culturing for a total of 21 days in Example 2 for transplantation for treatment of acute liver failure. DETAILED DESCRIPTION

[0047] The present application is described herein with reference to specific embodiments thereof which are illustrated in the accompanying drawings. The advantages and features of the present application will become apparent to those skilled in the art upon examination of the following details. Various modifications and changes can be made thereto without departing from the spirit and scope of the present application, which is set forth in the claims.

[0048] Example 1

[0049] The preparation of a regenerative decellularized liver scaffold (R-DLS) from an isolated liver organ of a healthy adult pig is as follows:

[0050] a. An isolated liver organ of a pig weighing about 20 kg and aged 15 weeks was taken.

[0051] b. The isolated liver organ was perfused with physiological saline having a mass fraction of 0.9% at a rate of 120 mL / min from the portal vein and the hepatic artery simultaneously for 30 min to wash out the red blood cells in the isolated liver organ.

[0052] c. The isolated liver organ treated in step b was perfused with sterile double-distilled water (ddH2O) at a rate of 120 mL / min from the portal vein and the hepatic artery simultaneously for 2 h to wash out the cellular components in the isolated liver organ.

[0053] d. The isolated liver organ treated in step c was perfused with a mixture of sodium dodecyl sulfate (SDS) having a mass fraction of 1% and a trace digestion solution (a mixture of trypsin having a mass fraction of 0.005% and ethylenediaminetetraacetic acid having a mass fraction of 0.002%) at a rate of 120 mL / min from the portal vein and the hepatic artery simultaneously for 72 h to wash out the cellular components in the isolated liver organ.

[0054] e. The perfusion of the isolated regenerating liver organ treated in step d is continued for 2 hours at a speed of 120 mL / min from the portal vein and the hepatic artery simultaneously with sterile double distilled water (ddH20) to wash out the mixture of sodium dodecyl sulfate and the trace digestion solution in the isolated regenerating liver organ;

[0055] f. The perfusion of the isolated regenerating liver organ treated in step e is continued for 1.5 hours at a speed of 120 mL / min from the portal vein and the hepatic artery simultaneously with 0.01 M phosphate buffer to wash out the sterile double distilled water in the isolated regenerating liver organ, restore the physiological state, and prepare a regenerating liver acellular biological scaffold (R-DLS). The perfusion process for preparing the R-DLS is shown in FIG. 1A, the pipeline system of the R-DLS is shown in FIG. IB, and the physical map of the R-DLS is shown in FIG. 1C. Figure 1 Figure 1 Figure 2

[0056] Example 2

[0057] Construction of a functional liver-like tissue in vitro using a hepatic oval cell line (HOCL)

[0058] (1) Preparation of a cell component-removed mammalian embryonic or neonatal liver filtrate

[0059] a. Under a dissecting microscope, liver-like tissues of 7 to 15 day embryonic mice (E7-E15) were taken out respectively, five embryonic mice per period, mixed, and then slowly ground using a homogenizer, filtered, and the filtrate was collected

[0060] b. The filtrate was repeatedly frozen and thawed at -80°C and 39°C for 3 times, each for 30 min, then filtered using a 0.45 μm filter membrane, the filtrate was collected, and a cell component-removed mouse embryonic liver filtrate was obtained;

[0061] c. The total protein content in the filtrate obtained in step b was detected using a protein kit, and was in the range of 50-100 mg / mL;

[0062] d. The filtrate obtained in step b was cultured under conventional cell culture conditions, and no cell growth was observed, indicating that the embryonic stem cells or progenitor cells in the filtrate were removed.

[0063] (2) Preparation of an endothelial cell differentiation-inducing culture medium, a bile duct cell differentiation-inducing culture medium, and a mature hepatocyte differentiation-inducing culture medium:

[0064] ​​​A. The cell component-removed mouse embryonic liver filtrate obtained in Example 2 was added to DMEM / F12 medium at a volume ratio of 1:12, and penicillin-G, streptomycin, L-glutamine, non-essential amino acids, sodium pyruvate and HEPES were added, and after mixing, bovine insulin, human transferrin, levothyroxine, triiodothyronine, sodium selenite, putrescine, progestin and albumin were added, to give a final concentration of 100 U / mL of penicillin-G, 100 μg / mL of streptomycin, 5 mM of L-glutamine, 1x of non-essential amino acids, 1x of sodium pyruvate, 25 mM of HEPES, 3.33 μg / mL of bovine insulin, 3.33 μg / mL of human transferrin, 0.26 μg / mL of levothyroxine, 0.22 μg / mL of triiodothyronine, 3.33 μg / mL of sodium selenite, 1.06 μg / mL of putrescine, 0.04 μg / mL of progestin and 0.04 μg / mL of albumin, to obtain a basic medium;

[0065] B. To the basic medium obtained in Step A, endothelial cell growth factor was added to give a final concentration of 20 ng / mL, to obtain an endothelial cell differentiation-inducing medium;

[0066] C. To the basic medium obtained in Step A, recombinant lgr5, recombinant HNF6 and anti-CEBP-β antibody were added to give a final concentration of 200 ng / mL of recombinant lgr5, 200 ng / mL of recombinant HNF6 and 50 ng / mL of anti-CEBP-β antibody, to obtain a bile duct cell differentiation-inducing medium;

[0067] D. To the basic medium obtained in Step A, recombinant HNF3β, recombinant HNF4α, anti-CEBP-α antibody, recombinant hepatocyte growth factor, recombinant beta-fibroblast growth factor, oncostatin M and dexamethasone were added to give a final concentration of 200 ng / mL of recombinant HNF3β, 200 ng / mL of recombinant HNF4α, 50 ng / mL of anti-CEBP-α antibody, 100 ng / mL of recombinant hepatocyte growth factor, 50 ng / mL of recombinant beta-fibroblast growth factor, 20 ng / mL of oncostatin M and 0.1 μM of dexamethasone, to obtain a mature hepatocyte differentiation-inducing medium.

[0068] (3) Construction of functional liver-like tissue in vitro

[0069] 1) 1.5 x 10 6Mesenchymal stem cells were implanted into the acellularized liver bioscaffold obtained in Example 1 through the portal vein, the hepatic artery and the inferior vena cava, respectively, and then placed in a 500 mL beaker, 400 mL of the induced endothelial cell differentiation culture solution prepared in step B was added, and static culture was carried out at 37°C and 5% CO2 for 7 days. The induced endothelial cell differentiation culture solution was added once a day for the first 4 days, and 10 mL was added each time. On the 5th day, half of the induced endothelial cell differentiation culture solution was replaced with the induced bile duct cell differentiation culture solution prepared in step C, and on the 6th day, the induced bile duct cell differentiation culture solution was completely replaced, and the culture was continued for another day. The liver-like tissue was obtained (i.e., the liver-like tissue was obtained after 7 days of total culture), as shown in FIG. 1B. Figure 2 As shown in FIG. 1B, the tissue showed a liver-like outline;

[0070] 2) 5 x 10 6 The liver oval cell line was implanted into the liver acellular bioscaffold treated in step 1) through the bile duct, and was first cultured statically for 3 days and then dynamically for 4 days at 37°C and 5% CO2. During the static culture, the induced bile duct cell differentiation culture solution was added once a day, and 10 mL was added each time. On the 1st day of dynamic culture, half of the induced bile duct cell differentiation culture solution was replaced with the induced mature hepatocyte differentiation culture solution prepared in step D (i.e., the volume ratio of induced bile duct cell differentiation culture solution to induced mature hepatocyte differentiation culture solution was 1:1), on the 2nd day, the volume ratio was 1:2, on the 3rd day, the volume ratio was 1:5, and on the 4th day, the induced mature hepatocyte differentiation culture solution was completely replaced. In the dynamic culture, the corresponding culture solution was input into the liver acellular bioscaffold through a circulating pump at 8:00 am-22:00 pm every day, and the culture was rotated at a speed of 10 rpm. The speed of the circulating pump was 20 rpm. In the static culture, the culture was statically cultured at 22:00 pm-8:00 am every day.

[0071] 3) The hepatic oval cell line was implanted into a decellularized liver scaffold treated in step 2) via the portal vein, hepatic artery, and inferior vena cava. The scaffold was then cultured at 37°C with alternating dynamic and static conditions for 11 days. During this period, 10 mL of inducing hepatocyte differentiation culture medium was added daily. Specifically, the dynamic culture was conducted from 8:00 AM to 10:00 PM daily, with the inducing hepatocyte differentiation culture medium being pumped into the decellularized liver scaffold while simultaneously rotating the scaffold horizontally at 10 rpm. The circulation pump speed was 20 rpm. The static culture was conducted from 10:00 PM to 8:00 AM daily. Liver-like tissue was obtained on day 7 (i.e., the total culture time was 21 days to obtain liver-like tissue). Figure 2 As shown in C.

[0072] During the static culture period in step 2), the ability of Ov6-positive hepatic oval cell lines to differentiate into CK19-positive bile duct cells was detected using immunofluorescence double staining. The results are as follows: Figure 3 As shown in A, by Figure 3 As shown in Figure A, hepatic oval cell lines are rapidly (approximately 2-24 hours) induced to differentiate into CK19-positive bile duct cells on a decellularized liver scaffold during the regeneration period. Figure 3 As indicated by the white arrow in section A;

[0073] During the alternating static and dynamic culture in step 3), the ability of Ov6-positive hepatic oval cell lines to differentiate into positive mature hepatocytes (ALBs) was detected using immunofluorescence double staining. The results are as follows: Figure 3 As shown in B, by Figure 3 As shown in the B diagram, hepatic oval cell lines are induced to differentiate into ALB on decellularized liver scaffolds during regeneration. Figure 3 As shown by the blue arrow in section B.

[0074] Example 3

[0075] Investigating the feasibility of constructing a functional liver tissue substitute using the method of this invention.

[0076] The supernatants of functional liver tissue cultures constructed at different stages (7d, 10d, 14d, 21d, 25d) in Example 2 were analyzed using Western blot technology. The results are as follows: Figure 4 As shown in A and B, by Figure 4 As shown in A and B, the expression of liver function proteins increases with the extension of culture time.

[0077] Liver function indicators of functional liver tissues constructed at different stages with different culture times (7d, 14d, 21d) were analyzed using a detection instrument. The results are as follows:Figure 4 As shown in Fig. C, wherein Fig. C is a diagram of total bilirubin and total bile acid test results in supernatant of each stage of functional hepatocyte-like tissue, respectively, b is a, and Figure 4 As shown in Fig. C, the functional hepatocyte-like tissue obtained by culturing for a total of 21 days can express functional proteins and secrete bile.

[0078] The functional hepatocyte-like tissue obtained by culturing for a total of 21 days in Example 2 was implanted into a 90% hepatectomy acute liver failure mouse model in a side-to-side anastomosis orthotopic auxiliary liver transplantation manner (experimental group), and a 90% hepatectomy acute liver failure mouse model was used as a control group, and the survival time of the two groups of mice was tested, and the results are shown in Fig. D. Figure 4 As shown in Fig. D, wherein Fig. D is a diagram of the survival time of the experimental group and the control group, and Figure 4 As shown in Fig. D, the survival time of the experimental group is significantly longer than that of the control group, indicating that the functional hepatocyte-like tissue constructed by the method has clinical transplantable potential.

[0079] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the present application, and they should be covered in the scope of the claims of the present application.

Claims

1. A method for inducing hepatic oval cell lines to form functional liver-like organ tissue on a decellularized liver scaffold, characterized in that, The method is as follows: (1) Mesenchymal stem cells were implanted into decellularized biological scaffolds of the liver via the portal vein, hepatic artery and inferior vena cava, respectively. Endothelial cell differentiation induction culture medium was added and statically cultured for 5-7 days. In the later stage of culture, the endothelial cell differentiation induction culture medium was gradually replaced with bile duct cell differentiation induction culture medium. The preparation methods of the culture medium for inducing endothelial cell differentiation and the culture medium for inducing bile duct cell differentiation are as follows: A. Add the homogenate filtrate of mammalian embryonic or neonatal liver (with cell components removed) to DMEM / F12 culture medium at a volume ratio of 1:4-32. Then add penicillin-G, streptomycin, L-glutamine, non-essential amino acids, sodium pyruvate, and HEPES. Mix well, then add bovine insulin, human transferrin, levothyroxine, triiodothyronine, sodium selenite, putrescine, progesterone, and albumin until the final concentration of penicillin-G is 100 U / mL, the final concentration of streptomycin is 100 μg / mL, and the final concentration of L-glutamine is... The final concentrations of non-essential amino acids, sodium pyruvate, and HEPES were all 25 mM. The final concentrations of bovine insulin, human transferrin, levothyroxine, triiodothyronine, sodium selenite, putrescine, progesterone, and albumin were 3.33 μg / mL, 0.26 μg / mL, 0.22 μg / mL, 3.33 μg / mL, 1.06 μg / mL, 0.04 μg / mL, and 0.04 μg / mL, respectively, were used to obtain the basal culture medium. B. Add endothelial growth factor to the basic culture medium obtained in step A until the final concentration of endothelial growth factor is 20 ng / mL to obtain an endothelial cell differentiation induction culture medium. C. Add recombinant lgr5, recombinant HNF6 and anti-CEBP-β antibody to the basic culture medium obtained in step A until the final concentration of recombinant lgr5 is 200 ng / mL, the final concentration of recombinant HNF6 is 200 ng / mL and the final concentration of anti-CEBP-β antibody is 50 ng / mL to obtain a culture medium for inducing bile duct cell differentiation. (2) The liver oval cell line was implanted into the decellularized liver biological scaffold after step (1) through the bile duct. It was first statically cultured for 3-5 days, and then dynamically cultured for 4-5 days. During the dynamic culture period, the bile duct cell differentiation induction culture medium was gradually replaced with the mature hepatocyte differentiation induction culture medium. The preparation method of the culture medium for inducing mature hepatocyte differentiation is as follows: Add recombinant HNF3β, recombinant HNF4α, anti-CEBP-α antibody, recombinant hepatocyte growth factor, recombinant beta-fibroblast growth factor, oncostatin M, and dexamethasone to the basal culture medium obtained in step A until the final concentrations of recombinant HNF3β, recombinant HNF4α, anti-CEBP-α antibody, recombinant hepatocyte growth factor, recombinant beta-fibroblast growth factor, oncostatin M, and dexamethasone are 20 ng / mL and 0.1 μM, respectively, to obtain a culture medium for inducing mature hepatocyte differentiation. (3) The liver oval cell line is implanted into the decellularized liver biological scaffold treated in step (2) through the portal vein, hepatic artery and inferior vena cava respectively, and cultured in alternating dynamic and static conditions for at least 7 days. The dynamic culture in the alternating dynamic and static culture is specifically as follows: under conditions of 37℃ and 5% CO2 (volume fraction), based on a culture container with a volume of 300-500 mL, the inducing mature hepatocyte differentiation culture medium is added once a day, 5-10 mL each time; from 8:00 am to 10:00 pm daily, the inducing mature hepatocyte differentiation culture medium is introduced into the liver decellularized biological scaffold through a circulation pump, while simultaneously rotating the culture horizontally at a speed of less than 20 rpm, the circulation pump speed being 20 rpm; the static culture in the alternating dynamic and static culture is specifically as follows: static culture from 10:00 pm to 8:00 am daily. The decellularized liver scaffold is a bioactive ex vivo decellularized liver scaffold in the regeneration phase.

2. The method as described in claim 1, characterized in that, In step A, the cellular-free mammalian embryonic or neonatal liver homogenate filtrate is prepared as follows: mammalian embryonic or neonatal liver tissue is taken, homogenized, filtered, and the filtrate is collected. Then, the filtrate is repeatedly frozen and thawed at least 3 times, each time for at least 30 minutes. Finally, the solid and liquid are separated, and the liquid is collected to obtain the cellular-free mammalian embryonic or neonatal liver homogenate filtrate.

3. The method as described in claim 1, characterized in that, In step (1), the amount of mesenchymal stem cells implanted is not less than 0.3-1.5 × 10⁻⁶ cells. 6 In step (2), the number of cells implanted into the hepatic oval cell line is not less than 1-5 × 10⁻⁶. 6 In step (3), the number of cells implanted into the hepatic oval cell line is not less than 0.3-1.5 × 10⁻⁶. 7 indivual.

4. The method according to any one of claims 1-3, characterized in that, The ex vivo regenerating liver is a mammalian ex vivo regenerating liver.

5. The method according to any one of claims 1-3, characterized in that, The method for preparing the bioactive ex vivo regenerating decellularized liver scaffold is as follows: (1) Take an isolated regenerating liver donor and wash out the red blood cells in the isolated regenerating liver by perfusion with physiological saline; (2) The cellular components in the liver treated in step (1) were dissolved and washed out by perfusion with sterile double-distilled water; (3) The cellular components in the liver treated in step (2) are washed out by perfusion using a mixture of sodium dodecyl sulfate and a micro-digestion solution; the micro-digestion solution is a mixture of trypsin and ethylenediaminetetraacetic acid. (4) The mixture of sodium dodecyl sulfate and trace amounts of digestive fluid in the liver treated in step (3) was washed out by perfusion with sterile double-distilled water; (5) Sterile double-distilled water in the liver treated in step (4) was washed out by perfusion with phosphate buffer solution to restore physiological state and obtain a bioactive ex vivo regenerated liver decellularized biological scaffold.

6. The method as described in claim 5, characterized in that, The method for preparing the bioactive ex vivo regenerating decellularized liver scaffold is as follows: (1) Take an isolated regenerated liver donor and perfuse the isolated regenerated liver with 0.9% physiological saline at a rate of 5-200 mL / min for 15-30 min to wash out the red blood cells in the isolated regenerated liver. (2) The liver treated in step (1) was perfused with sterile double-distilled water at a rate of 5-200 mL / min for 1-2 hours to wash out the cellular components in the ex vivo regeneration liver; (3) The liver treated in step (2) is perfused for 24-72 h at a rate of 5-200 mL / min with a mixture of sodium dodecyl sulfate (0.5-1% by mass) and a micro-digestion solution to wash out the cellular components in the ex vivo regeneration liver. The micro-digestion solution is a mixture of trypsin (0.0025-0.005% by mass) and ethylenediaminetetraacetic acid (0.001-0.002% by mass). (4) The liver treated in step (3) was perfused with sterile double-distilled water at a rate of 5-150 mL / min for 1-2 h to wash out the mixture of sodium dodecyl sulfate and trace digestive fluid in the ex vivo regeneration liver. (5) The liver treated in step (4) was perfused with 0.1-0.2M phosphate buffer at a rate of 5-150 mL / min for 1.5-2 h to wash out the sterile double-distilled water in the ex vivo regenerated liver, restore the physiological state, and obtain a decellularized biological scaffold of ex vivo regenerated liver with biological activity.

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