CN111979177B - Preparation method of human bile duct epithelial cells and culture medium thereof - Google Patents
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Abstract
The invention relates to a preparation method of human bile duct epithelial cells and a culture medium thereof, wherein the preparation method comprises the following steps: s1, separating hCMSCs by a homogenized tissue block method; s2, in-vitro directional differentiation of hCMCs into hepatic stem cells; s3, inducing hepatic stem cells to differentiate into bile duct epithelial cells. The invention provides a technical basis for clinically making biliary tract repair schemes and artificial bile duct engineering by inducing human chorionic mesenchymal stem cells (hCMSCs) to differentiate into bile duct epithelial cells in vitro. Experiments prove that the method can be used for preparing cells with physiological functions of bile duct epithelial cells.
Description
Technical Field
The invention belongs to the technical field of biological pharmacy, and particularly relates to a preparation method of human bile duct epithelial cells, a culture medium and a preparation method thereof.
Background
Clinically, the cause of injury to the biliary tract is complex, and iatrogenic biliary tract injury (Iatrogenic Bile Duct Injury, IBDI) accounts for about 95% of them. Iatrogenic biliary tract injury is injury to the biliary tract during abdominal surgery or biliary tract examination, resulting in damage to the integrity and patency of the biliary tract of a patient. Such as biliary tract system operation, pancreas operation, liver operation, gastrointestinal tract operation, etc. have the possibility of causing iatrogenic biliary tract injury, wherein bile duct injury caused by cholecystectomy operation accounts for more than 80%. In recent years, with the tremendous progress of laparoscopic techniques, LC is currently the first surgical type of cholecystectomy, but cannot effectively control the incidence of biliary tract injury. Some studies report that the incidence of laparoscopic cholecystectomy BDIs is between 0.4% and 0.7%, about one third of BDIs diagnosed in surgery and can be repaired or reconstructed immediately, and most BDIs are only found after surgery in clinical conditions such as abdominal pain, jaundice, bile peritonitis or shock.
The main cause of bile duct stenosis is excessive repair of scars. The related literature reports at home and abroad that the biliary tract scar formation mechanism can refer to the occurrence mechanism of skin scar healing, and the repairing mode can easily lead to bile duct stenosis (Benign Biliary Stricture, BBS). Then serious complications such as obstructive jaundice, recurrent cholangitis, intrahepatic duct stones, hepatic lobe/liver segment atrophy, biliary cirrhosis, portal hypertension and even liver failure appear, repeated operation treatment and even liver transplantation are needed, and huge pressure is brought to patients and families. Therefore, prevention and treatment of injured bile duct stenosis remains an important problem in biliary tract surgery, and how to treat bile duct stenosis after biliary tract injury is a problem that needs to be solved clinically.
Although the means for treating the stenosis after the bile duct injury and the materials for repairing the bile duct injury have more choices, the method has the limitation and the defects, particularly the traditional surgical anastomosis has more postoperative complications and repeated recurrence of the stenosis, so the method for continuously searching for the stenosis with small wound, quick recovery and low restenosis rate is urgent. If an artificial bile duct can be designed to replace the structure and even the function of the bile duct, the epithelial cells of the bile duct can be regenerated to repair the damage, and the method is a potential method.
Disclosure of Invention
The invention aims to provide a preparation method of human bile duct epithelial cells, a culture medium and a preparation method thereof, which provide a technical basis for clinically making biliary tract repair schemes and artificial bile duct engineering.
For this purpose, the invention provides a preparation method of human bile duct epithelial cells, which is characterized by comprising the following steps: s1, separating hCMSCs by a homogenized tissue block method; s2, in-vitro directional differentiation of hCMCs into hepatic stem cells; s3, inducing hepatic stem cells to differentiate into bile duct epithelial cells.
In some embodiments, the following features are also included:
the step 1 comprises the following steps: selecting placenta of term fetus, mechanically peeling placenta chorion tissue, repeatedly washing with PBS, and cutting chorion tissue into 0.8-1.2cm long strips; repeatedly washing with physiological saline to remove residual blood, and weighing; homogenizing to 0.1-0.3cm 3 Washing with physiological saline; centrifuging at 450-550r/min for 4-6min, removing supernatant, inoculating the precipitate into several culture dishes with a volume fraction of phi 100mm according to the amount of inoculating one culture dish with 4-6g placenta chorion tissue, and inverting the culture dishes with saturated humidity at 35-40deg.C4-6％CO 2 0.8-1.2h in the incubator. Then, the culture solution is gently added for in vitro culture: adding 2-8ng/ml bFGF human serum-free mesenchymal stem cell culture medium, placing at 35-40deg.C with 4% -6% CO 2 Culturing in an incubator, changing liquid once every 2-4 days, and passaging cells according to a conventional method after 12-14 days.
The steps 1 and 2 further comprise: hCMSCs were inoculated in six well plates: selecting a cell climbing sheet corresponding to a six-hole plate, soaking the cell climbing sheet in concentrated sulfuric acid overnight, washing the cell climbing sheet with tap water, soaking the cell climbing sheet in absolute alcohol for 5.5-6.5 hours, washing the cell climbing sheet with three distilled water for 2-4 times, putting the cell climbing sheet into an aluminum lunch box, drying the cell climbing sheet and sterilizing the cell climbing sheet under high pressure for later use; hCMSCs were cultured to passage 2, counted after 0.1% -0.25% pancreatin digestion and resuspended in medium. Before adding the cell suspension, a small amount of culture medium is added dropwise to each well according to the size of the slide, the slide and the bottom of the culture plate are bonded together, and then the culture plate is mixed with the culture medium at a ratio of 1-3×10 4 /cm 2 Concentration inoculation. Culturing in a saturated humidity incubator at 35-40deg.C and 4-6% CO 2.
The step S2 comprises the steps of flushing for 2-4 times by PBS (phosphate buffer solution) when the cell fusion rate in the six-hole plate reaches 65% -85%, adding a liver stem cell culture medium, and changing the liquid once every 3-4 days.
The liver stem cell culture medium comprises: human serum-free mesenchymal stem cell culture medium, 0.5% -1.5% of a mixture of green streptomycin, b FGF 5-15ng/ml, 0.5-2% of glutamine and 1-10 mug of hepatocyte growth factor.
The step S3 includes: and (3) after the cells of the six-hole plate are changed in an oval shape or a polygonal shape, discarding the old culture medium, flushing with PBS, adding the bile duct epithelial cell culture medium, changing the primary liquid every 3-4 days, and observing the morphological change of the cells under a microscope.
The cholangiocyte culture medium comprises: human serum-free mesenchymal stem cell culture medium, 0.5% -1.5% of a mixture of green streptomycin, 0.5% -2% of glutamine, 0.5% -2% of hepatocyte growth factor, 1% -5% of stem cell growth factor and 1% -10% of epidermal growth factor.
The invention also provides a liver cell culture medium, which comprises the following components: human serum-free mesenchymal stem cell culture medium, 0.5% -1.5% of a mixture of green streptomycin, b FGF 5-15ng/ml, 0.5-2% of glutamine and 1-10 mug of hepatocyte growth factor.
The invention also provides a bile duct epithelial cell culture medium, which is characterized by comprising the following components: serum-free mesenchymal stem cell culture medium, 0.5% -1.5% of a mixture of green streptomycin, 0.5% -2% of glutamine, 0.5% -2% of hepatocyte growth factor, 1% -5% of stem cell growth factor and 1% -10% of epidermal growth factor.
The invention provides a technical basis for clinically making biliary tract repair schemes and artificial bile duct engineering by inducing human chorionic mesenchymal stem cells (hCMSCs) to differentiate into bile duct epithelial cells in vitro. Experiments prove that the method can be used for preparing cells with physiological functions of bile duct epithelial cells.
Drawings
FIGS. 1A-1C are schematic illustrations of hCMCs under a primary and subculture microscope of hCMCs according to one embodiment of the invention.
FIGS. 2A-2E are graphs showing the results of flow cytometry hCMSCs phenotyping in accordance with one embodiment of the invention.
FIGS. 3A-3D are schematic diagrams of expression of hCMSCs vimentin and CK19 in accordance with one embodiment of the present invention.
FIGS. 4A-4B are schematic diagrams showing the results of the induction of differentiation of hCMCs into hepatic stem cells and identification of hCMCs in vitro into hepatoblasts according to one embodiment of the present invention.
FIGS. 5A-5B are graphs showing the differentiation and identification of hepatic stem cells into bile duct epithelial cells (induced bile duct epithelial cells and immunofluorescence) according to an embodiment of the present invention.
FIG. 6 is a schematic diagram of a technical route of one embodiment of the present invention.
Detailed Description
The primary task of biliary tract bioengineering is to find a stem cell with the capacity of differentiating into biliary tract epithelial cells, namely a bioengineered seed cell, which is induced to differentiate into biliary tract epithelial cells in vitro, so that the artificial bile duct has the structure and the function of the bile duct. In selecting stem cells, there are problems such as the differentiation and proliferation ability, difficulty in obtaining cells, and whether there is ethical dispute. At present, various tissue engineering researches on mesenchymal stem cells, such as amniotic mesenchymal stem cells, human umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells and the like, are more.
The bile duct epithelial cells are one layer of epithelial cells lined on the bile duct, maintain the integrity of the bile duct structure, and play an important role in bile secretion, anti-inflammation and the like. The literature reports that bile duct epithelial cells which grow in vivo and are freshly isolated are rectangular or columnar, a large amount of microvilli are arranged at the top of cells, cells cultured in vitro grow in an adherence manner, the cells are flattened to be in a paving stone-like epithelial cell form and are arranged into small 'cell islands', and the bile duct epithelial cells are differentiated and mature cells, so that the bile duct epithelial cells are easy to age, slow to grow and obviously lower in proliferation capacity after passage, and are difficult to culture. From the origin, liver oval cells have the capacity of bidirectional differentiation, and can be directionally differentiated into liver cells and bile duct epithelial cells. Researchers induced differentiation of hepatic oval cells into biliary epithelial cells in vitro; there have also been researchers that induce differentiation of bone marrow mesenchymal stem cells into hepatocytes, or indirectly or directly into bile duct epithelial cells.
The human chorionic mesenchymal stem cells have the characteristics of mesenchymal stem cells, have multidirectional differentiation potential, can be directionally differentiated into bile duct epithelial cells in vitro through induction, and can be used as potential sources of bile duct bioengineering cells. The following examples of the present invention provide a preparation scheme of human chorionic mesenchymal stem cells (Human Chorionic Mesenchymal Stem Cells, hCMSCs) and differentiation to bile duct epithelial cells by induction in vitro, which lay a foundation for bile duct bioengineering.
Compared with the scheme of inducing and differentiating the hepatic oval cells into bile duct epithelial cells in vitro and inducing and differentiating the bone marrow mesenchymal stem cells into the hepatic cells or indirectly or directly inducing and differentiating the bone marrow mesenchymal stem cells into the bile duct epithelial cells by other researchers, the method has the advantages that: hCMSCs are mesenchymal stem cells, are used as seed cells under various conditions, and are easy to separate and culture, wide in source, free of extra injury to human bodies, free of ethical disputes, strong in proliferation capacity, and strong in stem cell characteristics and multidirectional differentiation potential.
The basic method is as follows:
1) hCMCs are separated from placenta chorion tissue produced by term caesarean section by a homogenate tissue block method, the phenotype characteristics of hCMCs are identified by taking the 3 rd generation cells for flow cytometry detection, and the expression of hCMCs vimentin and CK-19 is detected by immunofluorescence staining.
2) In vitro directed differentiation of hCMSCs into hepatic stem cells: inoculating 3 rd generation hCMSCs into a 6-pore plate, adding into a liver stem cell culture medium, observing the growth and differentiation form of cells, and collecting cell culture liquid for biochemical detection of alkaline phosphatase, alpha fetoprotein and albumin content after the cells are changed in round, oval or polygonal shapes.
3) Inducing differentiation of hepatic stem cells into bile duct epithelial cells: and (3) adding a cholangiocellular epithelial cell culture medium into the induced cell 6 pore plate, observing the growth and differentiation conditions of cells, and detecting the expression of cytokeratin CK19 through immunofluorescence staining when the typical morphology of cholangiocellular is present, and identifying the differentiated cells.
Experiments prove that hCMSCs grow on the wall, cells are in a long spindle shape after passage and are arranged in a vortex shape, and the identification result of flow cytometry shows that hCMSCs express MSCs phenotype, vimentin and CK-19. The cells were observed to exhibit a typical oval or polygonal hepatic stem cell morphology by a contrast microscope after 2 weeks of induction and were arranged atypically in a nest. Compared with the prior induction, the hepatic stem cell marker alpha fetoprotein and albumin in the cell culture solution after induction are raised compared with the prior induction, the difference is statistically significant (p < 0.01), the mesenchymal stem cell marker alkaline phosphatase is reduced, and the difference is statistically significant (p < 0.01); after the cells continue to directionally induce differentiation for 3 weeks, the cells show morphological change of typical dendritic bile duct endothelial cells, and the induced cell immunity expression CK19 expression is up-regulated.
The specific method comprises the following steps:
hCMSCs in vitro separation and culture
Experimental materials
Fresh placenta of the term fetus of the healthy pregnant woman is obtained from the legal way, and the size and the weight are controlled within the normal range.
hCMSCs separation and in vitro amplification
1) Selecting placenta of term fetus, mechanically peeling placenta chorion tissue, repeatedly washing with PBS, and cutting chorion tissue into long strips of about 1 cm;
2) Repeatedly washing with physiological saline to remove residual blood, and weighing;
3) Homogenizing to 0.1-0.3cm 3 Washing with physiological saline.
4) Centrifuging at 450-550r/min for 4-6min, removing supernatant, inoculating the precipitate into several culture dishes with a volume fraction of phi 100mm according to the ratio of one culture dish for inoculating every 4-6g placenta chorion tissue, inverting the culture dishes at saturated humidity of 35-40deg.C and volume fraction of 4-6% CO 2 0.8-1.2h in the incubator. Then, the culture solution is gently added for in vitro culture: adding 2-8ng/ml bFGF human serum-free mesenchymal stem cell culture medium, placing at 35-40deg.C with 4% -6% CO 2 Culturing in an incubator, changing liquid once every 2-4 days, and passaging cells according to a conventional method after 12-14 days.
The morphological characteristics of hCMCs under a microscope are similar to the primary and passaged cell growth conditions, the cell expansion speed is slightly slower than that after P4 generation before P3 generation, and the multiplication speed of each generation of cells is basically stable along with the gradual purification of the cells.
Identification of biological characteristics of hCMSCs
1) Taking a human serum-free mesenchymal stem cell culture medium to culture cells after P3 generation, and stopping digestion by 0.125% -0.25% pancreatin/0.005% -0.02% EDTA;
2) DPBS is regulated to 4-6 multiplied by 10 5() Mu.l/40. Mu.l-60. Mu.l;
3) The mouse anti-human monoclonal antibodies CD73-PE, CD90-PE, CD105-PE, KDR-PE, CD14-PE, CD34-FITC, CD45-PE, CD105-PE and HLA-DR-PE are respectively added, the mixture is placed in a refrigerator at the temperature of 2-8 ℃ for reacting for 15-25min, and then washed twice by DPBS, and the cell phenotype is detected by a flow cytometer.
Detection result: according to the detection result of a flow cytometry, the P3 generation hCMSCs cultured in vitro under conventional culture conditions highly express CD73, CD90 and CD105, and do not express blood cell markers CD34, CD19, CD45 and HLA-DR, wherein the phenotype of the hCMSCs is the same as the surface markers proposed by the International placenta-derived stem cell conference.
3h hCMCs were inoculated in six well plates:
1) Preparing cell climbing tablet, namely selecting cell climbing tablet corresponding to six pore plates, soaking in concentrated sulfuric acid overnight, washing with tap water, soaking in absolute alcohol for 5.5-6.5 hours, washing with three distilled water for 2-4 times, drying in an aluminum lunch box, and sterilizing under high pressure for later use.
2) Cell seed plates: hCMSCs were cultured to passage 2, counted after 0.1% -0.25% pancreatin digestion and resuspended in medium. Before adding the cell suspension, a small amount of culture medium is added dropwise to each well according to the size of the slide, the slide and the bottom of the culture plate are bonded together, and then the culture plate is mixed with the culture medium at a ratio of 1-3×10 4 /cm 2 Concentration inoculation. Placing at 35-40deg.C and 4-6% CO 2 Culturing in a saturated humidity incubator.
Inducing hCMSCs to differentiate into liver stem cells and identifying:
after the cell fusion rate in the six-hole plate reaches 65-85%, PBS is used for washing for 2-4 times, and a liver-forming stem cell culture medium (human serum-free mesenchymal stem cell culture medium, 0.5-1.5% of a green streptomycin mixed solution, bFGF 5-15ng/ml, 0.5-2% of glutamine and 1-10 mu g of hepatocyte growth factor) is added, and the culture medium is changed once every 3-4 days. Observing the morphological change of the cells by an inverted phase contrast microscope, and taking cell culture fluid to biochemically detect the alkaline phosphatase, alpha fetoprotein and albumin levels after the cells are changed in oval shape or polygon shape.
The results show that the 3 rd generation hCMSCs differentiate into hepatic stem cells in vitro, after 2 weeks of induction by an inverted microscope, the cells are observed to gradually change from the original vortex-like arrangement to atypical nest-like arrangement under a low power microscope, and the cells are observed to gradually change from the original spindle shape or long spindle shape to an oval shape or a polygon shape under a high power microscope.
Inducing hepatic stem cells to differentiate into bile duct epithelial cells and identifying:
(1) Induction of bile duct forming epithelial cells: after the cells of the six-hole plate are changed in oval shape or polygonal shape, the old culture medium is discarded, PBS is used for washing, bile duct epithelial cell culture medium (human serum-free mesenchymal stem cell culture medium, 0.5% -1.5% of a mixture of green streptomycin, 0.5% -2% of glutamine, 0.5% -2% of hepatocyte growth factor, 1% -5% of stem cell growth factor and 1% -10% of epidermal cell growth factor) is added, the liquid is changed every 3-4 days, and the morphological change of the cells is observed under a microscope.
(2) Expression of cell CK-19 after immunofluorescence induction: and taking out the climbing sheet for staining when the cells are in a morphology similar to that of paving stone-like epithelial cells and the cells are subjected to cytoblast to cause the cells to present dendritic bile duct epithelial cell morphology change.
Staining a cell slide:
a. taking out the cell climbing tablet, and washing with PBS for 2-5 times, each time for 2-8min;
b. dropwise adding 2% -6% paraformaldehyde, fixing at 37deg.C for 25-35min, and washing with PBS for 2-5 times, each time for 2-8min;
c. dripping 0.1% -0.5% Triton-X100, penetrating membrane at 37deg.C for 10-20min, and washing with PBS for 2-8 times, each time for 2-8min;
e. dripping 10 mug/ml of immunofluorescence labeled CK-19 antibody to completely cover the specimen, and incubating for 0.5-1.5 h at 37 ℃ in a dark place;
f. taking out the slide, washing with PBS for 2-8 times for 2-8min each time, and then dying the cell nucleus with DAPI for 15-25min;
g. taking out the glass slide, washing the glass slide for 2 to 8 times by using PBS (phosphate buffer solution), and sucking redundant water by using filter paper, wherein the humidity of a specimen is kept;
h. immediately, photographs were observed under an inverted fluorescence microscope.
The results show that after the differentiation of hCMCs into liver stem cells is completed, the differentiation of liver stem cells into bile duct epithelial cells can be further induced.
Specific examples and experimental results are as follows:
example 1 in vitro isolation and culture of hCMSCs
Experimental materials
Fresh placenta of the term fetus of the healthy pregnant woman is obtained from the legal way, and the size and the weight are controlled within the normal range.
hCMSCs separation and in vitro amplification
1) Selecting placenta of term fetus, mechanically peeling placenta chorion tissue, repeatedly washing with PBS, and cutting chorion tissue into long strips of about 1 cm;
2) Repeatedly washing with physiological saline to remove residual blood, and weighing;
3) Homogenizing to 0.1-0.3cm 3 Washing with physiological saline.
4) Centrifuging at 500r/min for 5min, removing supernatant, inoculating the precipitate into several culture dishes with a volume fraction of 5% CO at 37deg.C and saturated humidity according to a fraction of phi 100mm culture dish inoculated per 5g placenta chorion tissue 2 For 1h in the incubator of (2). Then, the culture solution is gently added for in vitro culture: human serum-free mesenchymal stem cell culture medium added with 5ng/ml bFGF and placed at 37 ℃ and 5% CO 2 Culturing in an incubator, changing liquid once every 2-4 days, and passaging cells according to a conventional method after 12-14 days.
The morphological characteristics of hCMCs under a microscope are shown in figure 1, the growth conditions of the primary and the passage cells of the hCMCs are similar, flat monolayer cells are gradually formed after the primary cells are cultured for 2 weeks, the cells grow in a vortex shape, and along with the increase of the cell density, the cell bodies become slender, and the morphology is similar to that of fibroblast. The primary cells grow slowly, the bottle bottom is full of the primary cells for 3 to 4 weeks, the cultured cells can grow and passaged more stably, and the proliferation speed of the cells is not obviously slowed down after 10 generations of in vitro culture. The primary cells were the incubation period for growth 3-7 days after inoculation and culture, and the cells gradually began to adhere to the wall (FIG. 1A), without obvious expansion; after 7 days the cells entered the logarithmic phase, the proliferation of the cells was active and the division phase was seen, cells containing two nuclei appeared, the cell density was increased and connected to each other (FIG. 1B). Cell attachment can be observed under a phase contrast microscope 8 hours after cell passage, and after 3 days, the cells enter a logarithmic growth phase, and the amplification speed is obviously faster than that of primary culture (figure 1C); the bottom of the culture plate can be fully paved about 1 week. The cell expansion rate was slightly slower than after the P4 generation before the P3 generation, and the cell multiplication rate was substantially stable for each generation as the cells were gradually purified.
A great deal of research at home and abroad shows that bone marrow mesenchymal stem cells cultured in vitro can be successfully induced and differentiated into liver cells, and then small bile ducts are formed in a catchment area; there is also the induction of in vitro cultured bone marrow mesenchymal stem cells to differentiate directly into bile duct-like epithelial cells.
At presentThe mesenchymal stem cells are mainly bone marrow-derived, and have the advantages of convenience and rapidness, but have the defects: (1) the content of mesenchymal stem cells in human bone marrow is very small. Every 1×10 4 -1×10 5 The number of primary cells which can be obtained is limited, and the expansion and differentiation capacity and the cell number of the primary cells are obviously reduced along with the age; (2) the cell acquisition requires invasive operation, and the donor has uncomfortable feeling and is not easy to accept, and meanwhile, certain risk exists; (3) the chance of viral infection is great.
The placental chorionic mesenchymal stem cells can replace bone marrow mesenchymal stem cells, can make up the defects of the bone marrow mesenchymal stem cells, and have certain superiority. The placental chorionic mesenchymal stem cells have the following advantages: (1) the materials are hardly limited. Placenta is a "waste" which can be provided on the basis of informed consent of a healthy parturient who is normally delivered. The donor has no pain and less pollution; (2) the placental chorionic mesenchymal stem cells are more primitive and have lower immunogenicity, and the number of the obtained primary mesenchymal stem cells is large; (3) no further debate is related to social, ethical or legal aspects.
Therefore, chorionic mesenchymal stem cells are receiving a great deal of attention in the field of clinical transformation applications. However, the isolation and culture method of the placental chorionic mesenchymal stem cells is time-consuming and labor-consuming, the process is tedious and easy to pollute, and the isolation and culture method is an obstacle in the use of the chorionic mesenchymal stem cells.
At present, the chorionic mesenchymal stem cell separation and culture method mainly comprises a tissue block method and an enzyme digestion method, wherein the enzyme digestion method has the advantages of high cost, complex operation, large pollution opportunity, long enzyme digestion time, easiness in damaging cells and influence on the activity and passage of the cells. Tissue mass adherent culture is generally considered to be superior to trypsin digestion. The placenta leaflet is cut to 1mm by the traditional tissue block method 3 And spread into the culture dish at 1cm intervals, which consumes much time and labor.
The method is improved by selecting a tissue block method by using a handheld electric homogenizer, and (1) the method is easy to obtain, the basic function of the homogenizer is to be used for tissue homogenization, and biomedical laboratories commonly have the equipment. (2) The electric homogenizer is convenient to use, the equipment main body can be moved and sterilized at will, and the rotor of the electric homogenizer can be used for sterilizing. (3) The process is easy to grasp, the whole process is visible to naked eyes, and the treatment degree is adjusted at any time. In summary, the improved hCMCs separation culture method combines the advantages of tissue mass adherent culture methods using homogenizers. The improved technical system is simple, convenient and quick, improves the yield of the primary hCMSCs and lays a foundation for establishing a clinical hCMSCs library.
In addition, in the method, the chorion is processed to the size of rice grains by using a homogenizer, washing is carried out, most of fetal blood can be removed, and when the fetal blood is processed to a chylomorphic state, the fetal blood cells are preserved in supernatant by low-speed centrifugation again, so that the fetal blood cells are effectively removed, the influence of erythrocytes on the adhesion of hCMCs is reduced, and the uniformity of primary cells is improved. The whole process is simple and convenient, new reagents and treatment procedures are not needed to be added, and the pollution possibility is reduced.
The method improves the separation culture system of the placenta-derived chorionic mesenchymal stem cells on the basis of the traditional tissue block method, and has the advantages of simplicity, convenience, rapidness and easiness in obtaining a large number of primary placenta-derived chorionic mesenchymal stem cells in a short time.
Identification of biological characteristics of hCMSCs
1) Taking cells after P3 generation culture in a human serum-free mesenchymal stem cell culture medium, and stopping digestion by 0.25% pancreatin/0.01% EDTA;
2) DPBS is adjusted to 5 multiplied by 10 5 Mu.l/50;
3) The mouse anti-human monoclonal antibodies CD73-PE, CD90-PE, CD105-PE, KDR-PE, CD14-PE, CD34-FITC, CD45-PE, CD105-PE and HLA-DR-PE are respectively added, the mixture is placed in a refrigerator at 4 ℃ for reacting for 20min, and then washed twice by DPBS, and the cell phenotype is detected by a flow cytometer.
The detection results are shown in fig. 2: according to the detection result of the flow cytometry, the P3 generation hCMSCs cultured in vitro under the conventional culture conditions highly express CD73, CD90 and CD105, do not express blood cell markers CD34, CD19, CD45 and HLA-DR (figures 2A-E), and have the same phenotype as the surface markers proposed by the International placenta-derived stem cell conference.
Example 3h hCMSCs were seeded in six well plates:
1) Preparing cell climbing tablet, namely selecting cell climbing tablet corresponding to six pore plates, soaking in concentrated sulfuric acid overnight, washing with tap water, soaking in absolute alcohol for 6 hours, washing with three distilled water for 3 times, drying in an aluminum lunch box, and sterilizing under high pressure for later use.
2) Cell seed plates: hCMSCs were cultured to passage 2, counted after 0.125% pancreatin digestion and resuspended in medium. Before adding the cell suspension, a small amount of culture medium is added dropwise to each well according to the size of the slide, the slide and the bottom of the culture plate are bonded together, and then the culture plate is mixed with the culture medium at a ratio of 2×10 4 /cm 2 Concentration inoculation. Placing at 37deg.C and 5% CO 2 Culturing in a saturated humidity incubator.
Inducing hCMSCs to differentiate into liver stem cells and identifying:
after the cell fusion rate in the six-hole plate reaches about 70%, PBS is used for washing 3 times, a liver-forming stem cell culture medium (human serum-free mesenchymal stem cell culture medium, 1% of green streptomycin mixed solution, b FGF 10ng/ml, 1% of glutamine and 5 mug of hepatocyte growth factor) is added, and the culture medium is changed once every 3-4 days. Observing the morphological change of the cells by an inverted phase contrast microscope, and taking cell culture fluid to biochemically detect the alkaline phosphatase, alpha fetoprotein and albumin levels after the cells are changed in oval shape or polygon shape.
The factor combination can induce the bone marrow mesenchymal stem cells cultured in vitro to differentiate into liver stem cells and promote the proliferation of the liver stem cells, and the cells have the dual functions of the bone marrow mesenchymal stem cells and the liver stem cells. However, a culture scheme of adding foetal calf serum or serum replacement to a basic culture medium such as DMEM is generally adopted, and a human serum-free mesenchymal stem cell culture medium is selected, so that the introduction of heterologous proteins is avoided, and a proper amount of factor combination is added, so that the culture method is more suitable for the culture of human hepatogenic stem cells.
As a result, as shown in FIG. 4, the 3 rd generation hCMSCs differentiated in vitro into hepatic stem cells, and after 2 weeks of induction, the cells were observed to gradually change from the original vortex arrangement to an atypical nest arrangement under a low power microscope, and from the original spindle shape or long spindle shape to an oval shape or a polygonal shape under a high power microscope (see FIGS. 4A-B). The results of alkaline phosphatase, alpha fetoprotein and albumin detection in the cell culture broth before and after induction are shown in Table 1.
Table 1: alkaline phosphatase, alpha fetoprotein and albumin levels in cell culture media before and after induced differentiation of hCMSCs to hepatic stem cells
And (3) table notes: compared with the prior induction, the P is less than 0.01,
P＜0.01，/>
P＜0.01/>
example 5 induction of hepatic stem cell differentiation into bile duct epithelial cells and identification:
induction of bile duct forming epithelial cells: after the cells of the six-hole plate are changed in oval shape or polygonal shape, discarding the old culture medium, flushing with PBS, adding bile duct epithelial cell culture medium (human serum-free mesenchymal stem cell culture medium, 1% of green streptomycin mixed solution, 1% of glutamine, 1% of hepatocyte growth factor, 2% of stem cell growth factor and 5% of epidermal cell growth factor), changing the primary liquid every 3-4 days, and observing the cell morphology change under a microscope.
Less research is carried out on the in vitro cultured bile duct epithelial cells, and the adult bile duct epithelial cells are difficult to separate and purify, are used as differentiated and mature cells, are easy to age in the in vitro culture, have reduced proliferation capacity in the culture process, have shorter in vitro maintenance time (3-4 weeks at maximum), and can have the phenomena of mutation, degeneration, death and the like.
Common culture media for culturing bile duct epithelial cells include DMEM, DMEM/F12, RPMI-1640, BDCM and the like. Different media were selected according to the purpose of the study, and the corresponding additional ingredients were added. Some growth factors, hormones and amino acids are used as additives to promote proliferation of bile duct epithelial cells. Comprises keratin cell growth factors, diffusion factors, hepatocyte growth factors and the like, and has obvious proliferation promoting effect on bile duct epithelial cells.
The method uses the hCMCs to form bile duct epithelial cells by a two-part induction method through improving a culture medium formula, the hCMCs are convenient to take materials, the separation and purification of the hCMCs are easy to operate by an improved homogenate tissue block method, and the hCMCs are used as an undifferentiated mature stem cell to induce the bile duct epithelial cells to be cultured in vitro so as to be not easy to age, the proliferation capacity is stable in the culture process, the in vitro maintenance time is long, and the aging phenomenon and the like can not occur in a short time.
Expression of cell CK-19 after immunofluorescence induction: and taking out the climbing sheet for staining when the cells are in a morphology similar to that of paving stone-like epithelial cells and the cells are subjected to cytoblast to cause the cells to present dendritic bile duct epithelial cell morphology change.
Staining a cell slide:
a. taking out the cell climbing sheet, and cleaning the cell climbing sheet with PBS liquid for 3 times, each time for 5min;
b. dropwise adding 4% paraformaldehyde, fixing at 37deg.C for 30min, and washing with PBS for 3 times each for 5min;
c. dropwise adding 0.3% Triton-X100, penetrating through the membrane at 37deg.C for 15min, and washing with PBS for 3 times each for 5min;
e. dripping 10 mug/ml of immunofluorescence labeled CK-19 antibody to completely cover the specimen, and incubating for 1h at 37 ℃ in dark;
f. taking out the slide, washing 3 (2-8) times with PBS for 5min each time, and then dying the cell nucleus with DAPI for 20min;
g. taking out the glass slide, flushing the glass slide for 3 times by using PBS, and sucking redundant water by using filter paper, wherein the humidity of the sample is kept;
h. immediately, photographs were observed under an inverted fluorescence microscope.
As shown in FIG. 5, after the completion of the differentiation of hCMCs into liver stem cells, the differentiation of liver stem cells into bile duct epithelial cells was further induced, and after 21 days of the induction culture, the cells were gradually changed from the original oval shape and polygonal shape into quasi-circular shape, and the cell processes were observed, and a stringiness phenomenon was observed between the cells, so that small "cell islands" were formed by cell arrangement, and the morphology was similar to that of epithelial cells and was in the form of a paving stone (see FIG. 5A). The expression of CK19 in the induced cells was detected by immunofluorescence, and CK19 positive cells were seen under immunofluorescence microscope, and the expression of some cells was strongly positive, and "pseudopodia" like projections were seen (see FIG. 5B). The cells were shown to have physiological functions of bile duct epithelial cells.
The hCMSCs used in the method of the embodiment of the invention are adult stem cells with mesenchymal sources, are used as various conditions of seed cells, are easy to separate and culture, have wide sources, have no extra damage to human bodies and no ethical dispute, have strong proliferation capacity, and have stronger stem cell characteristics and multidirectional differentiation potential; the stem cells cultured by each placenta chorion can be infused by about 2 ten thousand people, thereby meeting the industrial preparation.
Claims (8)
1. The preparation method of the human bile duct epithelial cells is characterized by comprising the following steps:
s1, separating hCMSCs by a homogenized tissue block method;
s2, in-vitro directional differentiation of hCMCs into hepatic stem cells;
s3, inducing hepatic stem cells to differentiate into bile duct epithelial cells.
2. The method for preparing human bile duct epithelial cells according to claim 1, wherein said step S1 comprises: selecting placenta of term fetus, mechanically peeling placenta chorion tissue, repeatedly washing with PBS, and cutting chorion tissue into 0.8-1.2cm long strips; repeatedly washing with physiological saline to remove residual blood, and weighing; homogenizing to 0.1-0.3cm 3 Washing with physiological saline; centrifuging at 450-550r/min for 4-6min, removing supernatant, inoculating the precipitate into several culture dishes with a volume fraction of 4-6% CO at 35-40deg.C and saturated humidity according to the amount of one culture dish with a diameter of 100mm inoculated into every 4-6g placenta chorion tissue 2 0.8-1.2h in the incubator. Then, the culture solution is gently added for in vitro culture: human none supplemented with 2-8ng/ml bFGFSerum mesenchymal stem cell culture medium is placed at 35-40 ℃ and 4% -6% CO 2 Culturing in an incubator, changing liquid once every 2-4 days, and passaging cells according to a conventional method after 12-14 days.
3. The method for preparing human bile duct epithelial cells according to claim 2, wherein between the step S1 and the step S2, further comprises: hCMSCs were inoculated in six well plates: selecting a cell climbing sheet corresponding to a six-hole plate, soaking the cell climbing sheet in concentrated sulfuric acid overnight, washing the cell climbing sheet with tap water, soaking the cell climbing sheet in absolute alcohol for 5.5-6.5 hours, washing the cell climbing sheet with three distilled water for 2-4 times, putting the cell climbing sheet into an aluminum lunch box, drying the cell climbing sheet and sterilizing the cell climbing sheet under high pressure for later use; hCMSCs were cultured to passage 2, counted after 0.1% -0.25% pancreatin digestion and resuspended in medium. Before adding the cell suspension, a small amount of culture medium is added dropwise to each well according to the size of the slide, the slide and the bottom of the culture plate are bonded together, and then the culture plate is mixed with the culture medium at a ratio of 1-3×10 4 /cm 2 Concentration inoculation. Placing at 35-40deg.C and 4-6% CO 2 Culturing in a saturated humidity incubator.
4. The method for preparing human bile duct epithelial cells according to claim 1, wherein the step S2 comprises washing the cells in the six-well plate with PBS for 2-4 times until the cell fusion rate reaches 65% -85%, adding into the culture medium of hepatic stem cells, and changing the liquid once every 3-4 days.
5. The method of preparing human bile duct epithelial cells according to claim 1, wherein said liver blast stem cell culture medium comprises: human serum-free mesenchymal stem cell culture medium, 0.5% -1.5% of a mixture of green streptomycin, b FGF 5-15ng/ml, 0.5-2% of glutamine and 1-10 mug of hepatocyte growth factor.
6. The method for preparing human bile duct epithelial cells according to claim 5, wherein said step S3 comprises: and (3) after the cells of the six-hole plate are changed in an oval shape or a polygonal shape, discarding the old culture medium, flushing with PBS, adding the bile duct epithelial cell culture medium, changing the primary liquid every 3-4 days, and observing the morphological change of the cells under a microscope.
7. The method of preparing human bile duct epithelial cells according to claim 6, wherein said bile duct epithelial cell culture medium comprises: human serum-free mesenchymal stem cell culture medium, 0.5% -1.5% of a mixture of green streptomycin, 0.5% -2% of glutamine, 0.5% -2% of hepatocyte growth factor, 1% -5% of stem cell growth factor and 1% -10% of epidermal growth factor.
8. A liver stem cell culture medium, comprising: human serum-free mesenchymal stem cell culture medium, 0.5% -1.5% of a mixture of green streptomycin, b FGF 5-15ng/ml, 0.5-2% of glutamine and 1-10 mug of hepatocyte growth factor, wherein the hepatoblast stem cell culture medium is used for inducing bone marrow mesenchymal stem cells cultured in vitro to differentiate into hepatic stem cells.
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