CN110669721A - Method for inducing hepatic oval cell line to form functional hepatic organ-like tissue on liver decellularization biological scaffold - Google Patents
Method for inducing hepatic oval cell line to form functional hepatic organ-like tissue on liver decellularization biological scaffold Download PDFInfo
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Abstract
The invention relates to a method for inducing a Hepatic Oval Cell Line (HOCL) to form a functional hepatic sample organ-like tissue on a liver decellularization biological scaffold, which belongs to the technical field of biology. In the method, HOCL of an in-vitro 3D culture system in R-DLS shows that the HOCL is differentiated to functional liver cells within 2-24 hours, a liver-like organ tissue can be formed within 21 days, and the new liver constructed by the method has important clinical conversion value for serving as a liver transplantation donor and treating ESLD.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for inducing hepatic oval cell lines to form functional hepatic organ-like tissues on a liver decellularization biological scaffold.
Background
End-stage Liver diseases (ESLD) including Acute Liver failure (Acute Liver failure) and Chronic Liver cirrhosis (Chronic Liver cirrhosis) have high mortality rate, seriously harm human health and are also serious diseases in China. The only effective treatment method at present is liver transplantation, but the liver transplantation is difficult to be widely applied due to donor deficiency. Therefore, finding an effective alternative to liver function is the key to solving this problem. At present, although various harmful substances generated or increased due to liver failure are removed by means of an in vitro temporary auxiliary or replacement method of an artificial liver such as a liver, for example, a Bioartificial liver (BAL), the symptoms of a patient in an advanced stage are only temporarily improved, and thus, the complete replacement of liver functions cannot be realized all the time. In recent years, studies on the engineering of organoid tissues using seed cells and normal organ skeletons have been more extensive, but there are fewer liver organs having biological activity successfully constructed in vitro. The in vitro construction of organ tissues by using organ skeletons and seed cells mainly faces three difficulties: (1) optimizing the bracket material; (2) selecting seed cells; (3) and (3) establishing a microenvironment for in vitro 3D culture.
Scaffolds for liver tissue reconstruction mainly include biological scaffolds and physical scaffolds. The physical stent is made of a material with good biocompatibility into the shape of the liver stent, but the existing 3D printing technology cannot construct an accurate liver complex pipeline (such as a portal vein, an artery affected and a bile duct) system. The biological scaffold adopts a natural liver scaffold, completely retains a liver pipeline system, and becomes the mainstream direction of the current research, but the biological scaffold also needs to ensure the physiological activity when the cell-removing activity treatment is carried out, so that the clinically realizable method and the result report for successfully constructing the liver tissue by using the biological scaffold are less.
The seed cells for liver construction need to have the potential of differentiating into hepatocytes and cholangiocytes, and adult hepatocytes and Mesenchymal Stem Cells (MSCs) are adopted in the academic circles at present, wherein the application of the cells is greatly limited because the in vitro long-term culture of the hepatocytes is poor in survival condition in the existing method and the MSCs lack the potential of largely differentiating into functional hepatocytes.
The in vitro microenvironment is mainly established by establishing a conditioned medium and culture conditions simulating the liver growth environment, wherein the research on the culture medium is mainly focused on the culture of liver cells at present, and the culture medium for constructing liver tissues is rarely reported.
CN104894066A discloses a method for in vitro 3-D culture and reconstruction of artificial liver by taking stem cell group NG2+ HSC as seed cells, but the method uses hepatic stem cells/progenitor cells (NG2+ HSC) expressing neural-glial antigen 2 as seed cells, the seed cells are complex in preparation method and not beneficial to large-scale use, and in the process of in vitro reconstruction of artificial liver by (NG2+ HSC), three different culture media need to be replaced, the operation process is complex and not easy to control, the time for reconstruction of artificial liver is long, and the method is not beneficial to large-scale application.
The liver oval cell line (HOCL) is a liver stem cell with multi-differentiation potential, can be activated to differentiate into hepatocytes, cholangiocytes and other histiocytes under certain conditions, and can be obtained by commercial means, so that the liver oval cell line not only has convenient source, but also can be quickly differentiated into a large number of mature hepatocytes, cholangiocytes and functional liver tissue structures (about 1-24h), and the stronger functional differentiation potential lays a theoretical foundation for constructing functional liver tissues in vitro.
Disclosure of Invention
In view of the above, the present invention provides a method for inducing hepatic oval cell lines to form functional hepatic organ-like tissue on a liver decellularization biological scaffold.
In order to achieve the purpose, the invention provides the following technical scheme:
1. a method of inducing hepatic oval cell lines to form functional hepatic organoid tissue on a liver decellularization bioscaffold, the method comprising:
(1) respectively implanting mesenchymal stem cells into a liver acellular biological scaffold through a portal vein, a hepatic artery and an inferior vena cava, adding an induced endothelial cell differentiation culture solution, 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;
(2) implanting a hepatic oval cell line into the liver acellular biological scaffold treated in the step (1) through a bile duct, performing static culture for 3-5 days, performing dynamic culture for 4-5 days, and gradually replacing the culture solution for inducing bile duct cell differentiation with culture solution for inducing mature hepatic cell differentiation during the dynamic culture period;
(3) and (3) respectively implanting the hepatic oval cell line into the liver acellular biological scaffold treated in the step (2) through the portal vein, the hepatic artery and the inferior vena cava, and performing dynamic and static alternate culture for at least 7 days.
Preferably, the preparation methods of the endothelial cell differentiation induction culture solution, the cholangiocyte differentiation induction culture solution and the mature hepatocyte differentiation induction culture solution are as follows:
A. adding the homogenate filtrate of the liver of the mammal in the embryonic stage or the newborn stage without cell components into DMEM/F12 culture solution according to the volume ratio of 1:4-32, then adding penicillin-G, streptomycin, L-glutamine, unnecessary amino acids, sodium pyruvate and HEPES, evenly mixing, then adding bovine insulin, human transferrin, levothyroxine, triiodothyroxine, sodium selenite, putrescine, progestational hormone and albumin until the final concentration of the penicillin-G is 100U/mL, the final concentration of the streptomycin is 100 mu G/mL, the final concentration of the L-glutamine is 5mM, 1X unnecessary amino acids, 1X sodium pyruvate, the final concentration of the HEPES is 25mM, the final concentration of the bovine insulin is 3.33 mu G/mL, the final concentration of the human transferrin is 3.33 mu G/mL, the final concentration of the levothyroxine is 0.26 mu G/mL, the final concentration of triiodothyroxine is 0.22 mug/mL, the final concentration of sodium selenite is 3.33 mug/mL, the final concentration of putrescine is 1.06 mug/mL, the final concentration of progestogen is 0.04 mug/mL, and the final concentration of albumin is 0.04 mug/mL, so as to obtain a basic culture solution;
B. adding endothelial cell growth factors into the basic culture solution obtained in the step A until the final concentration of the endothelial cell growth factors is 20ng/mL, and obtaining an endothelial cell differentiation induction culture solution;
C. adding recombinant lgr5, recombinant HNF6 and anti-CEBP-beta antibody into the basic culture solution obtained in the step A until the final concentration of the recombinant lgr5 is 200ng/mL, the final concentration of the recombinant HNF6 is 200ng/mL and the final concentration of the anti-CEBP-beta antibody is 50ng/mL to obtain a culture solution for inducing the differentiation of the cholangiocytes;
D. adding recombinant HNF into the basic culture solution obtained in the step A3Beta, recombinant HNF4 alpha, anti-CEBP-alpha antibody, recombinant hepatocyte growth factor, recombinant beta-fiber growth factor, oncostatin M and dexamethasone to the recombinant HNF3The final concentration of beta is 200ng/mL, the final concentration of recombinant HNF4 alpha is 200ng/mL, the final concentration of anti-CEBP-alpha antibody is 50ng/mL, the final concentration of recombinant hepatocyte growth factor is 100ng/mL, the final concentration of recombinant beta-fiber growth factor is 50ng/mL, the final concentration of oncostatin M is 20ng/mL, and the final concentration of dexamethasone is 0.1 mu M, thus obtaining the culture solution for inducing the differentiation of mature hepatocytes.
Preferably, in step a, the cell-free liver homogenate filtrate in the embryonic stage or the neonatal stage of the mammal is prepared as follows: homogenizing liver tissue of mammal embryo stage or newborn stage, filtering, collecting filtrate, repeatedly freezing and thawing the filtrate for at least 3 times (each time at least 30 mm), solid-liquid separating, and collecting liquid to obtain homogenized liver filtrate of mammal embryo stage or newborn stage with cell components removed.
Preferably, the first and second liquid crystal materials are,
in the step (1), the volume fraction of CO is 5 percent at 37 DEG C2Statically culturing for 5-7 days under the condition, supplementing the induced endothelial cell differentiation culture solution for 1 time every day in the first 3-5 days according to a culture container with the volume of 300-500mL, wherein 5-10mL of the induced endothelial cell differentiation culture solution is added every time, half of the induced endothelial cell differentiation culture solution is replaced by the induced cholangiocyte differentiation culture solution in the 3-5 days, and the induced cholangiocyte differentiation culture solution is completely replaced in the 4-6 days;
in the step (2), the volume fraction of CO is 5% at 37 DEG C2Under the condition, firstly statically culturing for 3-5 days, then dynamically culturing for 4-5 days, wherein in the static culture period, the culture solution for inducing the differentiation of the bile duct cells is supplemented for 1 time every day according to a culture container with the volume of 300-500mL, and each time is 5-10 mL; replacing half of the cholangiocyte-inducing differentiation culture solution with mature hepatocyte-inducing differentiation culture solution on the 1 st day of the dynamic culture, gradually increasing the replacement amount of the mature hepatocyte-inducing differentiation culture solution on the following days, and completely replacing with the mature hepatocyte-inducing differentiation culture solution on the last day;
in the step (3), the volume fraction of CO is 5% at 37 DEG C2Performing dynamic and static alternate culture under the condition, wherein the culture medium for inducing the mature hepatic cells to differentiate is supplemented for 1 time every day in the period of 5-10mL each time according to a culture container with the volume of 300-500 mL.
Preferably, the dynamic culture in the dynamic and static alternate culture specifically comprises: inputting a culture solution for inducing mature hepatic cell differentiation into a liver acellular biological scaffold by a circulating pump at a speed of 8:00am-22:00pm every day, and horizontally rotating and culturing at a rotating speed of less than 20rpm, wherein the speed of the circulating pump is 20 rpm; the static culture in the dynamic and static alternate culture specifically comprises the following steps: static culture was performed at 22:00pm-8:00am per day.
Preferably, in the step (1), the mesenchymal stem cells are implanted in an amount of not less than 0.3 to 1.5X 106A plurality of; in the step (2), the cell amount implanted by the hepatic oval cell line is not less than 1-5 multiplied by 106A plurality of; in the step (3), the cell amount implanted by the hepatic oval cell line is not less than 0.3-1.5 multiplied by 107And (4) respectively.
Preferably, the liver decellularization biological scaffold is an ex vivo regeneration-period liver decellularization biological scaffold with biological activity.
Preferably, the ex vivo liver is a mammalian ex vivo liver.
Preferably, the preparation method of the in vitro regeneration period liver decellularization biological scaffold with biological activity comprises the following steps:
(1) taking an in vitro regeneration-period liver donor, and washing out red blood cells in the in vitro regeneration-period liver by adopting a physiological saline in a perfusion mode;
(2) dissolving and washing out cell components in the liver treated in the step (1) by adopting sterile double distilled water in a perfusion mode;
(3) washing out the cell components in the liver treated in the step (2) by adopting a mixed solution of sodium dodecyl sulfate and trace digestive juice in a perfusion mode; the micro digestive juice is a mixed solution of trypsin and ethylenediamine tetraacetic acid;
(4) washing out the mixed solution of the sodium dodecyl sulfate and the trace digestive juice in the liver treated in the step (3) by adopting sterile double distilled water in a perfusion mode;
(5) and (4) washing out the sterile double distilled water in the liver treated in the step (4) by adopting phosphate buffer saline solution in a perfusion mode, and recovering the physiological state to prepare the in vitro regeneration period liver acellular biological scaffold with biological activity.
Preferably, the preparation method of the in vitro regeneration period liver decellularization biological scaffold with biological activity comprises the following steps:
(1) taking an in vitro regeneration-period liver donor, and perfusing the in vitro regeneration-period liver for 15-30min at the speed of 5-200mL/min by adopting 0.9% of physiological saline in mass fraction so as to wash out red blood cells in the in vitro regeneration-period liver;
(2) perfusing the liver treated in the step (1) with sterile double distilled water at a speed of 5-200mL/min for 1-2h to wash out cell components in the liver in the in vitro regeneration period;
(3) continuously perfusing the liver treated in the step (2) for 24-72h at the speed of 5-200mL/min by adopting a mixed solution of 0.5-1% by mass of sodium dodecyl sulfate and a trace digestive juice to wash out cell components in the liver in the in vitro regeneration period, wherein the trace digestive juice is a mixed solution of 0.0025-0.005% by mass of trypsin and 0.001-0.002% by mass of ethylenediaminetetraacetic acid;
(4) continuously perfusing the liver treated in the step (3) for 1-2h at the speed of 5-150mL/min by adopting sterile double distilled water so as to wash out the mixed liquid of the sodium dodecyl sulfate and the trace digestive juice in the liver in the in vitro regeneration period;
(5) and (3) continuously infusing the liver treated in the step (4) for 1.5-2h at the speed of 5-150mL/min by adopting 0.1-0.2M phosphate buffer solution to wash out the sterile double distilled water in the liver in the in vitro regeneration period, recovering the physiological state and preparing the in vitro regeneration period liver acellular biological scaffold with biological activity.
The invention has the beneficial effects that: the invention provides a method for inducing a hepatic oval cell line to form a functional hepatic sample organ-like tissue on a liver decellularization biological scaffold, which mainly takes a Hepatic Oval Cell Line (HOCL) as a seed stem cell, implants the stem cell into a regeneration-stage liver decellularization biological scaffold (R-DLS) with bioactivity, adds a conditional culture solution capable of inducing the HOCL liver to be functionally differentiated and reconstructing a functional hepatic organ tissue structure according to the concept of simulating a somatic hepatic organ development microenvironment in vitro, and constructs the functional hepatic sample organ-like tissue in a 3D culture system in vitro. The method overcomes the defect that the artificial material in the existing 3D printing technology can not construct complex vital organs such as liver, has convenient source, simple operation, low cost and short induction culture time, shows that the HOCL of an in-vitro 3D culture system in R-DLS differentiates to functional liver cells within 2-24h, and can form a liver-like organ tissue within 21 days, so that the 'new liver' constructed by the method has important clinical conversion value for serving as a liver transplantation donor and treating ESLD.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention. The objectives and other advantages of the invention may be realized and attained by the means of the instrumentalities and combinations particularly pointed out hereinafter.
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For the purposes of promoting a better understanding of the objects, aspects and advantages of the invention, reference will now be made to the following detailed description taken in conjunction with the accompanying drawings in which:
FIG. 1 is a diagram of perfusion process and the piping system of the in vitro regeneration phase hepatocyte-removed biological scaffold prepared from ex vivo regeneration phase liver organ of pig in example 1; (in FIG. 1, A is a perfusion process diagram of the in vitro regeneration period liver organ acellular biological scaffold prepared from the ex vivo regeneration period liver organ of pig in example 1; and in FIG. 1, B is a system diagram of each pipeline of the in vitro regeneration period liver organ acellular biological scaffold)
FIG. 2 is a diagram of decellularized biological entities of liver organs in an ex vivo regeneration phase and a diagram of liver-like tissues formed in each time period of culture, which are used in the construction of functional liver-like tissues in vitro in example 2; (FIG. 2A is a schematic diagram showing the in vitro regeneration period of the decellularized biological scaffold for liver organ in example 1; FIG. 2B is a schematic diagram showing the appearance of the liver-like tissue after the total culture period of 7 days; FIG. 2C is a schematic diagram showing the appearance of the liver-like tissue after the total culture period of 21 days)
FIG. 3 is a graph showing the results of the potency test that the liver oval cell line stained positively by Ov6 was induced to differentiate into CK 19-positive cholangiocytes and positive mature hepatocytes, respectively, in the 3D culture system of the present invention in example 2; (FIG. 3A is a graph showing the results of measurement of the ability of inducing the hepatic oval cell line stained positively by Ov6 to differentiate into the bile duct cells positive by CK19 by immunofluorescence double staining during the static culture in step 2) in the in vitro construction of functional liver-like tissue in example 2; FIG. 3B is a graph showing the results of the immunofluorescence double staining assay for the differentiation of the Ocv 6-stained hepatic oval cell line into positive mature hepatocytes (ALB) during the alternate culture at step 3) under an active-static condition
FIG. 4 is a graph showing the results of tests on secretion of hepatic function proteins and transplantation therapy of acute liver failure in the supernatant of the in vitro constructed functional liver-like tissue culture in example 2 (in FIG. 4, A is a graph showing the results of detection of bile duct cell proteins positive to CK19 in the supernatant of each functional liver-like tissue culture, B is a graph showing the results of detection of mature hepatic cell proteins positive to the supernatant of each functional liver-like tissue culture, A and B are graphs showing the results of tests on total bilirubin and total bile acid in the supernatant of each functional liver-like tissue culture, respectively, in FIG. 4, and D is a graph showing the results of the transplantation therapy of acute liver failure of functional liver-like tissue obtained in example 2 with a total culture period of 21 days in example 4).
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Example 1
The preparation of the in vitro regeneration period Liver organ acellular biological Scaffold (R-DLS) of the healthy adult pig comprises the following specific steps:
a. taking in-vitro liver organs of pigs with the weight of about 20kg and the age of 15 weeks in a regeneration period;
b. perfusing the liver organ in the in vitro regeneration phase from the portal vein and the hepatic artery simultaneously for 30min at a speed of 120mL/min by using 0.9% of physiological saline in mass fraction to wash out red blood cells in the liver organ in the in vitro regeneration phase;
c. sterile double distilled water (ddH) is adopted2O) perfusing the ex vivo regenerative liver organ treated in the step b from the portal vein and the hepatic artery at the same time for 2h at a speed of 120mL/min to wash out cellular components in the ex vivo regenerative liver organ;
d. c, continuously perfusing the liver organ in the in vitro regeneration phase treated in the step c from the portal vein and the hepatic artery at the same time for 72 hours by adopting a mixed solution of Sodium Dodecyl Sulfate (SDS) with the mass fraction of 1% and a trace digestive juice (a mixed solution of trypsin with the mass fraction of 0.005% and ethylenediamine tetraacetic acid with the mass fraction of 0.002%) at the speed of 120mL/min to wash out cell components in the liver organ in the in vitro regeneration phase;
e. sterile double distilled water (ddH) is adopted2O) continuously perfusing the liver organ in the ex vivo regeneration period treated in the step d from the portal vein and the hepatic artery for 2h at the same time at a speed of 120mL/min to wash out the mixed solution of sodium dodecyl sulfate and trace digestive juice in the liver organ in the ex vivo regeneration period;
f. and (3) continuously perfusing the liver organ in the isolated regeneration period treated in the step (e) from the portal vein and the hepatic artery by using 0.01M phosphate buffer solution at the speed of 120mL/min for 1.5h to wash out sterile double distilled water in the liver organ in the isolated regeneration period, recovering the physiological state and preparing a biological scaffold for decellularizing the liver in the regeneration period (R-DLS), wherein the perfusion process for preparing the R-DLS is shown as A in the figure 1, the pipeline systems of the R-DLS are shown as B in the figure 1, and the physical diagram of the R-DLS is shown as A in the figure 2.
Example 2
In vitro construction of functional liver-like tissue using Hepatic Oval Cell Line (HOCL)
(1) Preparing cell-free liver filtrate of mammal at embryonic stage or newborn stage
a. Respectively taking liver-like tissues of 7-15 day embryonic mice (E7-E15) under dissecting microscope, mixing five embryonic mice at each stage, slowly grinding with homogenizer, filtering, and collecting filtrate
b. Repeatedly freezing and thawing the filtrate at-80 deg.C and 39 deg.C for 3 times (30 mm each time), filtering with 0.45 μm filter membrane, and collecting filtrate to obtain mouse embryo stage liver filtrate with cell components removed;
c. b, detecting that the total protein content in the filtrate obtained in the step b is within the range of 50-100mg/mL by using a protein kit;
d. culturing the filtrate obtained in step b under conventional cell culture conditions and finding no evidence of cell growth, demonstrating that the embryonic stem cells or progenitor cells in the filtrate have been removed.
(2) Preparing an endothelial cell differentiation induction culture solution, a bile duct cell differentiation induction culture solution and a mature hepatocyte differentiation induction culture solution:
A. adding the liver filtrate obtained in example 2 after removing the cell components into DMEM/F12 culture solution according to the volume ratio of 1:12, adding penicillin-G, streptomycin, L-glutamine, unnecessary amino acids, sodium pyruvate and HEPES, uniformly mixing, adding bovine insulin, human transferrin, levothyroxine, triiodothyroxine, sodium selenite, putrescine, progestogen and albumin until the final concentration of penicillin-G is 100U/mL, the final concentration of streptomycin is 100 mu G/mL, the final concentration of L-glutamine is 5mM, 1X unnecessary amino acids, 1X sodium pyruvate, the final concentration of HEPES is 25mM, the final concentration of bovine insulin is 3.33 mu G/mL, the final concentration of human transferrin is 3.33 mu G/mL, the final concentration of levothyroxine is 0.26 mu G/mL, the final concentration of triiodothyroxine is 0.22 mu G/mL, the final concentration of sodium selenite is 3.33 mug/mL, the final concentration of putrescine is 1.06 mug/mL, the final concentration of progestogen is 0.04 mug/mL, and the final concentration of albumin is 0.04 mug/mL, so as to obtain a basic culture solution;
B. adding endothelial cell growth factors into the basic culture solution obtained in the step A until the final concentration of the endothelial cell growth factors is 20ng/mL, and obtaining an endothelial cell differentiation induction culture solution;
C. adding recombinant lgr5, recombinant HNF6 and anti-CEBP-beta antibody into the basic culture solution obtained in the step A until the final concentration of recombinant lgr5 is 200ng/mL, the final concentration of recombinant HNF6 is 200ng/mL and the final concentration of anti-CEBP-beta antibody is 50ng/mL to obtain a culture solution for inducing the differentiation of the cholangiocytes;
D. adding recombinant HNF into the basic culture solution obtained in the step A3Beta, recombinant HNF4 alpha, anti-CEBP-alpha antibody, recombinant hepatocyte growth factor, recombinant beta-fiber growth factor, oncostatin M and dexamethasone to recombinant HNF3The final concentration of beta is 200ng/mL, the final concentration of recombinant HNF4 alpha is 200ng/mL, the final concentration of anti-CEBP-alpha antibody is 50ng/mL, the final concentration of recombinant hepatocyte growth factor is 100ng/mL, the final concentration of recombinant beta-fiber growth factor is 50ng/mL, the final concentration of oncostatin M is 20ng/mL, and the final concentration of dexamethasone is 0.1 mu M, thus obtaining the culture solution for inducing the differentiation of mature hepatocytes.
(3) In vitro construction of functional liver-like tissue
1) Mixing 1.5X 106The mesenchymal stem cells were implanted into the liver decellularized biological scaffold obtained in example 1 through the portal vein, hepatic artery and inferior vena cava, respectively, and then placed into a 500mL beaker, and 400mL of the culture solution for inducing endothelial cell differentiation prepared in step B was added thereto, and CO was added thereto at 37 ℃ in a volume fraction of 5%2Performing static culture for 7 days under the condition, supplementing the induced endothelial cell differentiation culture solution for 1 time every day in the first 4 days, wherein each time is 10mL, replacing half of the induced endothelial cell differentiation culture solution with the induced cholangiocyte differentiation culture solution prepared in the step C on the 5 th day, completely replacing the induced cholangiocyte differentiation culture solution on the 6 th day, and continuing to culture for 1 day to obtain a liver-like tissue (namely, obtaining the liver-like tissue after the total culture time is 7 days), wherein as shown in B in figure 2, the liver-like contour of the tissue is known to appear;
2) will be 5X 106Implanting individual hepatic oval cell lines into the liver decellularized biological scaffold treated in the step 1) through a bile duct, and carrying out CO treatment at 37 ℃ and with the volume fraction of 5%2Under the condition, firstly statically culturing for 3 days, then dynamically culturing for 4 days, and replenishing the culture solution for inducing the differentiation of the bile duct cells for 1 time every day in the static culture period, wherein 10mL of the culture solution is added each time; replacing half of the cholangiocyte-inducing differentiation culture solution with the mature hepatocyte-inducing differentiation culture solution prepared in the step D on the 1 st day of dynamic culture (namely, replacing the cholangiocyte-inducing differentiation culture solution with the mature hepatocyte-inducing differentiation culture solution according to the volume ratio of 1:1), replacing the cholangiocyte-inducing differentiation culture solution with the mature hepatocyte-inducing differentiation culture solution according to the volume ratio of 1:2 on the 2 nd day, replacing the cholangiocyte-inducing differentiation culture solution with the mature hepatocyte-inducing differentiation culture solution according to the volume ratio of 1:5 on the 3 rd day, and completely replacing the cholangiocyte-inducing differentiation culture solution with the mature hepatocyte-inducing differentiation culture solution on the 4 th day; wherein, the dynamic culture in the dynamic and static alternate culture specifically comprises the following steps: inputting corresponding culture solution into the liver acellular biological scaffold by a circulating pump at a speed of 10rpm for horizontal rotation culture at 8:00am-22:00pm every day, wherein the speed of the circulating pump is 20rpm, and the static culture in the dynamic and static alternate culture specifically comprises the following steps: standing and culturing at 22:00pm-8:00am every day;
3) implanting the hepatic oval cell line into the liver decellularized biological scaffold treated in the step 2) through the portal vein, the hepatic artery and the inferior vena cava, and performing CO treatment at 37 ℃ and with the volume fraction of 5%2Performing dynamic and static alternate culture for 11 days under the condition, and supplementing an induced mature hepatocyte differentiation culture solution for 1 time every day, wherein each time is 10mL, and the dynamic culture in the dynamic and static alternate culture specifically comprises the following steps: inputting a culture solution for inducing mature hepatic cells to a liver acellular biological scaffold by a circulating pump at a speed of 10rpm for horizontal rotary culture at 8:00am-22:00pm every day, wherein the speed of the circulating pump is 20rpm, and the static culture in the dynamic and static alternate culture specifically comprises the following steps: the liver-like tissue was obtained by static culture at 22:00pm-8:00am per day until day 7 (i.e., the total length of culture was 21 days), as shown in C in FIG. 2.
During the static culture period in the step 2), the immunofluorescence double staining method is adopted to detect the capability of inducing the hepatic oval cell line stained positive by Ov6 to differentiate into the bile duct cells which are CK19 positive, the detection result is shown as A in figure 3, and as can be seen from A in figure 3, the hepatic oval cell line is rapidly induced and differentiated into the bile duct cells which are CK19 positive on the liver decellularization biological scaffold in the regeneration period (about 2-24h), and is shown as a white arrow in figure 3A;
during the dynamic and static alternate culture period in the step 3), the immunofluorescence double staining method is adopted to detect the capability of inducing the hepatic oval cell line with positive Ov6 staining to be differentiated into positive mature hepatic cells (ALB), the detection result is shown as B in figure 3, and as can be seen from B in figure 3, the hepatic oval cell line is induced to be differentiated into ALB on the liver decellularized biological scaffold in the regeneration stage, as is shown by blue arrows in B in figure 3.
Example 3
Explore the feasibility of functional liver-like tissue function replacement constructed by the method of the invention
The functional hepatic tissue-like culture supernatants of the respective stages constructed in example 2 at different culture times (7d, 10d, 14d, 21d, and 25d) were examined by western blot technique, and as shown in A and B in FIG. 4, it was found that hepatic function protein expression increased with the increase of the culture time from A and B in FIG. 4.
Analyzing the liver function indexes of the functional liver-like tissues at each stage constructed at different culture time (7d, 14d and 21d) by using a detector, wherein the result is shown as C in figure 4, wherein a and b in C are respectively a test result graph of total bilirubin and total bile acid in the supernatant of the functional liver-like tissues at each stage, and as can be seen from C in figure 4, the functional liver-like tissues obtained within the total culture time of 21 days can perform functional protein expression and bile secretion.
The functional liver tissue obtained in the total culture time of 21 days in the example 2 is implanted into a 90% hepatectomy acute liver failure mouse model (experimental group) in a mode of end-to-side anastomosis and in-situ auxiliary liver transplantation, meanwhile, the 90% hepatectomy acute liver failure mouse model is used as a control group, the survival time of two groups of mice is tested, the result is shown as D in figure 4, and the survival time of the experimental group is obviously prolonged compared with that of the control group according to D in figure 4, which shows that the functional liver tissue constructed by the method has clinical transplantable potential.
Finally, the above embodiments are only intended to illustrate the technical solutions of the present invention and not to limit the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions, and all of them should be covered by the claims of the present invention.
Claims (10)
1. A method of inducing hepatic oval cell lines to form functional hepatic organoid tissue on a liver decellularization bioscaffold, the method comprising:
(1) respectively implanting mesenchymal stem cells into a liver acellular biological scaffold through a portal vein, a hepatic artery and an inferior vena cava, adding an induced endothelial cell differentiation culture solution, 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;
(2) implanting a hepatic oval cell line into the liver acellular biological scaffold treated in the step (1) through a bile duct, performing static culture for 3-5 days, performing dynamic culture for 4-5 days, and gradually replacing the culture solution for inducing bile duct cell differentiation with culture solution for inducing mature hepatic cell differentiation during the dynamic culture period;
(3) and (3) respectively implanting the hepatic oval cell line into the liver acellular biological scaffold treated in the step (2) through the portal vein, the hepatic artery and the inferior vena cava, and performing dynamic and static alternate culture for at least 7 days.
2. The method according to claim 1, wherein the endothelial cell differentiation-inducing culture solution, the cholangiocyte differentiation-inducing culture solution and the mature hepatocyte differentiation-inducing culture solution are prepared by:
A. adding the homogenate filtrate of the liver of the mammal in the embryonic stage or the newborn stage without cell components into DMEM/F12 culture solution according to the volume ratio of 1:4-32, then adding penicillin-G, streptomycin, L-glutamine, unnecessary amino acids, sodium pyruvate and HEPES, evenly mixing, then adding bovine insulin, human transferrin, levothyroxine, triiodothyroxine, sodium selenite, putrescine, progestational hormone and albumin until the final concentration of the penicillin-G is 100U/mL, the final concentration of the streptomycin is 100 mu G/mL, the final concentration of the L-glutamine is 5mM, 1X unnecessary amino acids, 1X sodium pyruvate, the final concentration of the HEPES is 25mM, the final concentration of the bovine insulin is 3.33 mu G/mL, the final concentration of the human transferrin is 3.33 mu G/mL, the final concentration of the levothyroxine is 0.26 mu G/mL, the final concentration of triiodothyroxine is 0.22 mug/mL, the final concentration of sodium selenite is 3.33 mug/mL, the final concentration of putrescine is 1.06 mug/mL, the final concentration of progestogen is 0.04 mug/mL, and the final concentration of albumin is 0.04 mug/mL, so as to obtain a basic culture solution;
B. adding endothelial cell growth factors into the basic culture solution obtained in the step A until the final concentration of the endothelial cell growth factors is 20ng/mL, and obtaining an endothelial cell differentiation induction culture solution;
C. adding recombinant lgr5, recombinant HNF6 and anti-CEBP-beta antibody into the basic culture solution obtained in the step A until the final concentration of the recombinant lgr5 is 200ng/mL, the final concentration of the recombinant HNF6 is 200ng/mL and the final concentration of the anti-CEBP-beta antibody is 50ng/mL to obtain a culture solution for inducing the differentiation of the cholangiocytes;
D. adding recombinant HNF into the basic culture solution obtained in the step A3Beta, recombinant HNF4 alpha, anti-CEBP-alpha antibody, recombinant hepatocyte growth factor, recombinant beta-fiber growth factor, oncostatin M and dexamethasone to the recombinant HNF3Final concentration of beta200ng/mL, the final concentration of the recombinant HNF4 alpha is 200ng/mL, the final concentration of the anti-CEBP-alpha antibody is 50ng/mL, the final concentration of the recombinant hepatocyte growth factor is 100ng/mL, the final concentration of the recombinant beta-fiber growth factor is 50ng/mL, the final concentration of the oncostatin M is 20ng/mL, and the final concentration of dexamethasone is 0.1 mu M, so as to obtain the culture solution for inducing the differentiation of the mature hepatocytes.
3. The method of claim 2, wherein in step a, the cell-depleted mammalian fetal-stage or neonatal liver homogenate filtrate is prepared by: homogenizing liver tissue of mammal embryo stage or newborn stage, filtering, collecting filtrate, repeatedly freezing and thawing the filtrate for at least 3 times (each time at least 30 mm), solid-liquid separating, and collecting liquid to obtain homogenized liver filtrate of mammal embryo stage or newborn stage with cell components removed.
4. The method of claim 1,
in the step (1), the volume fraction of CO is 5 percent at 37 DEG C2Statically culturing for 5-7 days under the condition, supplementing the induced endothelial cell differentiation culture solution for 1 time every day in the first 3-5 days according to a culture container with the volume of 300-500mL, wherein 5-10mL of the induced endothelial cell differentiation culture solution is added every time, half of the induced endothelial cell differentiation culture solution is replaced by the induced cholangiocyte differentiation culture solution in the 3-5 days, and the induced cholangiocyte differentiation culture solution is completely replaced in the 4-6 days;
in the step (2), the volume fraction of CO is 5% at 37 DEG C2Under the condition, firstly statically culturing for 3-5 days, then dynamically culturing for 4-5 days, wherein in the static culture period, the culture solution for inducing the differentiation of the bile duct cells is supplemented for 1 time every day according to a culture container with the volume of 300-500mL, and each time is 5-10 mL; replacing half of the cholangiocyte-inducing differentiation culture solution with mature hepatocyte-inducing differentiation culture solution on the 1 st day of the dynamic culture, gradually increasing the replacement amount of the mature hepatocyte-inducing differentiation culture solution on the following days, and completely replacing with the mature hepatocyte-inducing differentiation culture solution on the last day;
in the step (3), the volume fraction of CO is 5% at 37 DEG C2Dynamic and static state under the conditionAlternately culturing, and supplementing the culture solution for inducing the differentiation of the mature hepatocytes for 1 time and 5-10mL each time according to a culture container with the volume of 300-500 mL.
5. The method of claim 4, wherein the dynamic culture in the dynamic and static alternate culture is specifically: inputting a culture solution for inducing mature hepatic cell differentiation into a liver acellular biological scaffold by a circulating pump at a speed of 8:00am-22:00pm every day, and horizontally rotating and culturing at a rotating speed of less than 20rpm, wherein the speed of the circulating pump is 20 rpm; the static culture in the dynamic and static alternate culture specifically comprises the following steps: static culture was performed at 22:00pm-8:00am per day.
6. The method of claim 1, wherein the mesenchymal stem cell is implanted in the amount of not less than 0.3-1.5 x 10 in step (1)6A plurality of; in the step (2), the cell amount implanted by the hepatic oval cell line is not less than 1-5 multiplied by 106A plurality of; in the step (3), the cell amount implanted by the hepatic oval cell line is not less than 0.3-1.5 multiplied by 107And (4) respectively.
7. The method of any one of claims 1 to 6, wherein the liver decellularized biological scaffold is a biologically active ex vivo regenerative liver decellularized biological scaffold.
8. The method of claim 7, wherein the ex vivo liver for regeneration is a mammalian ex vivo liver for regeneration.
9. The method of claim 7, wherein the bioactive ex vivo liver decellularization bioscaffold is prepared by:
(1) taking an in vitro regeneration-period liver donor, and washing out red blood cells in the in vitro regeneration-period liver by adopting a physiological saline in a perfusion mode;
(2) dissolving and washing out cell components in the liver treated in the step (1) by adopting sterile double distilled water in a perfusion mode;
(3) washing out the cell components in the liver treated in the step (2) by adopting a mixed solution of sodium dodecyl sulfate and trace digestive juice in a perfusion mode; the micro digestive juice is a mixed solution of trypsin and ethylenediamine tetraacetic acid;
(4) washing out the mixed solution of the sodium dodecyl sulfate and the trace digestive juice in the liver treated in the step (3) by adopting sterile double distilled water in a perfusion mode;
(5) and (4) washing out the sterile double distilled water in the liver treated in the step (4) by adopting phosphate buffer saline solution in a perfusion mode, and recovering the physiological state to prepare the in vitro regeneration period liver acellular biological scaffold with biological activity.
10. The method of claim 9, wherein the bioactive ex vivo liver decellularization bioscaffold is prepared by:
(1) taking an in vitro regeneration-period liver donor, and perfusing the in vitro regeneration-period liver for 15-30min at the speed of 5-200mL/min by adopting 0.9% of physiological saline in mass fraction so as to wash out red blood cells in the in vitro regeneration-period liver;
(2) perfusing the liver treated in the step (1) with sterile double distilled water at a speed of 5-200mL/min for 1-2h to wash out cell components in the liver in the in vitro regeneration period;
(3) continuously perfusing the liver treated in the step (2) for 24-72h at the speed of 5-200mL/min by adopting a mixed solution of 0.5-1% by mass of sodium dodecyl sulfate and a trace digestive juice to wash out cell components in the liver in the in vitro regeneration period, wherein the trace digestive juice is a mixed solution of 0.0025-0.005% by mass of trypsin and 0.001-0.002% by mass of ethylenediaminetetraacetic acid;
(4) continuously perfusing the liver treated in the step (3) for 1-2h at the speed of 5-150mL/min by adopting sterile double distilled water so as to wash out the mixed liquid of the sodium dodecyl sulfate and the trace digestive juice in the liver in the in vitro regeneration period;
(5) and (3) continuously infusing the liver treated in the step (4) for 1.5-2h at the speed of 5-150mL/min by adopting 0.1-0.2M phosphate buffer solution to wash out the sterile double distilled water in the liver in the in vitro regeneration period, recovering the physiological state and preparing the in vitro regeneration period liver acellular biological scaffold with biological activity.
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