CN111979177A - Preparation method and culture medium of human bile duct epithelial cells - Google Patents
Preparation method and culture medium of human bile duct epithelial cells Download PDFInfo
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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 hMSCs by a homogenate tissue block method; s2, directionally differentiating the hCSs in vitro into hepatic stem cells; and S3, inducing the hepatic stem cells to differentiate into bile duct epithelial cells. The invention provides a technical basis for clinically establishing a biliary tract repair scheme and an artificial bile duct engineering by inducing human chorionic mesenchymal stem cells (hCSCs) to differentiate into bile duct epithelial cells in vitro. Experiments prove that the method can be used for preparing the cells with the physiological function of the 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 of the human bile duct epithelial cells.
Background
Clinically, the cause of biliary tract Injury is complicated, and Iatrogenic biliary tract Injury (IBDI) accounts for about 95% of the biliary tract injuries. Iatrogenic biliary tract injury is injury to the bile duct during abdominal surgery or during biliary tract examination, which results in the integrity and patency of the patient's biliary tract being destroyed. Such as biliary tract system operation, pancreas operation, liver operation, gastrointestinal tract operation and the like, iatrogenic biliary tract injury is possible, wherein biliary duct injury caused by cholecystectomy accounts for more than 80%. In recent years, with the great progress of laparoscopic technology, LC is the first choice for cholecystectomy, but the incidence of biliary tract injury cannot be effectively controlled. Some studies report that the incidence of laparoscopic cholecystectomy BDI is between 0.4% and 0.7%, approximately one-third of BDI is diagnosed during surgery and can be immediately repaired or rebuilt, and most BDI are only detected after surgery in clinical signs, such as abdominal pain, jaundice, biliary peritonitis, or shock.
The main cause of biliary stricture is excessive repair of scar. The related literature reports at home and abroad that the Biliary tract scar formation mechanism can refer to the generation mechanism of skin scar healing, and the repair mode can easily cause bile duct stenosis (BBS). Serious complications such as obstructive jaundice, recurrent cholangitis, intrahepatic bile duct calculi, liver lobe/liver segment atrophy, biliary cirrhosis, portal hypertension, even liver failure and the like occur subsequently, repeated surgical treatment and even liver transplantation are needed, and great pressure is brought to patients and families. Therefore, prevention and treatment of the damaged biliary stricture are still important problems in biliary tract surgery, and how to treat the biliary stricture after the biliary tract injury is a problem to be solved urgently in clinic.
Although the means for treating the stenosis after the bile duct injury and the materials for repairing the bile duct injury are selected more, the method has the limitations and the disadvantages, especially the traditional surgical anastomosis has more postoperative complications and repeated recurrence of the stenosis, so that the continuous search for a method 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 method is a method with development potential for regenerating bile duct epithelial cells to repair the injury.
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, and provides a technical basis for clinically establishing a biliary tract repair scheme and an artificial bile duct engineering.
Therefore, the invention provides 1a preparation method of human bile duct epithelial cells, which is characterized by comprising the following steps: s1, separating hMSCs by a homogenate tissue block method; s2, directionally differentiating the hCSs in vitro into hepatic stem cells; and S3, inducing the 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 a full-term fetus, mechanically stripping chorion tissues of the placenta, repeatedly washing by PBS, and shearing the chorion tissues into long blocks of 0.8-1.2 cm; repeatedly washing with normal saline to remove residual blood, and weighing; homogenizing to 0.1-0.3cm3Washing with physiological saline; centrifuging at 450-2The incubator for 0.8-1.2 h. Then culture solution is added gently for in vitro culture: adding 2-8ng/ml bFGF human serum-free mesenchymal stem cell culture medium, placing at 35-40 deg.C and 4-6% CO2Culturing in an incubator, changing the culture solution once every 2-4 days, and carrying out passage on the cells according to a conventional method after 12-14 days.
The method also comprises the following steps between the steps 1 and 2: hCMSCs were seeded in six-well plates: selecting cell climbing sheets corresponding to a six-hole plate, soaking in concentrated sulfuric acid overnight, washing with tap water, soaking in anhydrous alcohol for 5.5-6.5 hours, washing with triple-distilled water for 2-4 times, drying in an aluminum lunch box, and sterilizing under high pressure for later use; hCMSCs were cultured to passage 2, counted after 0.1% -0.25% trypsinization and resuspended in culture medium. Before adding cell suspension, small amount of culture medium is dropped into each well according to the size of the slide to adhere the slide and the bottom of the culture plate together, and then the amount of culture medium is 1-3 × 104/cm2And (4) concentration inoculation. Culturing in an incubator with 35-40 deg.C, 4-6% CO2 and saturated humidity.
And the step S2 comprises the steps of flushing 2-4 times with PBS when the cell fusion rate in the six-hole plate reaches 65% -85%, adding the adult liver stem cell culture medium, and changing the culture medium every 3-4 days.
The culture medium for the liver forming stem cells comprises: human serum-free mesenchymal stem cell culture medium, 0.5-1.5% streptomycin mixed solution, 5-15ng/ml of b FGF, 0.5-2% glutamine and 1-10 mu g of hepatocyte growth factor.
The step S3 includes: and when the cells of the six-hole plate are changed into an oval shape or a polygonal shape, abandoning the old culture medium, washing with PBS, adding the cholangioblast forming epithelial cell culture medium, changing the medium every 3-4 days, and observing the morphological change of the cells under a microscope.
The cholangioblast epithelial cell culture medium comprises: human serum-free mesenchymal stem cell culture medium, 0.5-1.5% streptomycin mixed solution, 0.5-2% glutamine, 0.5-2% hepatocyte growth factor, 1-5% stem cell growth factor and 1-10% epidermal growth factor.
The invention also provides a hepatoblast culture medium, which comprises: human serum-free mesenchymal stem cell culture medium, 0.5-1.5% streptomycin mixed solution, 5-15ng/ml of b FGF, 0.5-2% glutamine and 1-10 mu g of hepatocyte growth factor.
The invention also provides a cholangioblast epithelial cell culture medium, which is characterized by comprising the following components: serum-free mesenchymal stem cell culture medium, 0.5-1.5% streptomycin mixed solution, 0.5-2% glutamine, 0.5-2% hepatocyte growth factor, 1-5% stem cell growth factor and 1-10% epidermal growth factor.
The invention provides a technical basis for clinically establishing a biliary tract repair scheme and an artificial bile duct engineering by inducing human chorionic mesenchymal stem cells (hCSCs) to differentiate into bile duct epithelial cells in vitro. Experiments prove that the method can be used for preparing the cells with the physiological function of the bile duct epithelial cells.
Drawings
FIGS. 1A-1C are schematic diagrams of hCMSCs under a primary and subculture microscope, according to one embodiment of the present invention.
FIGS. 2A-2E are graphs showing the results of phenotypic analysis of flow cytometry hCMSCs according to one embodiment of the present invention.
Figures 3A-3D are schematic representations of the expression of hCMSCs vimentin and CK19, according to one embodiment of the invention.
FIGS. 4A-4B are schematic diagrams of the results of inducing differentiation of hCMSCs into hepatic stem cells and their identification (differentiation of hCMSCs into hepatocytes in vitro) according to an embodiment of the present invention.
FIGS. 5A-5B are graphs showing the results of inducing differentiation of hepatic stem cells into biliary epithelia and identifying the induced biliary epithelia (induced biliary epithelia and immunofluorescence) according to an embodiment of the present invention.
FIG. 6 is a schematic diagram of a technical route of an embodiment of the present invention.
Detailed Description
The first task of biliary tract bioengineering is to find one kind of stem cell capable of differentiating into biliary epithelial cell, i.e. bioengineering seed cell, and induce and differentiate in vitro into biliary epithelial cell to make the artificial bile duct possess the structure and function of bile duct. When selecting stem cells, the differentiation and proliferation ability, the ease of obtaining cells, and the presence or absence of ethical disputes need to be considered. At present, various tissue engineering researches 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.
The bile duct epithelial cells are a layer of epithelial cells lining the bile duct, maintain the structural integrity of the bile duct, and play an important role in bile secretion, inflammation resistance and the like of the bile duct. The related literature reports that bile duct epithelial cells grown in vivo and separated freshly are rectangular or columnar, a large amount of microvilli are arranged on the tops of the cells, the cells cultured in vitro grow in an adherence manner, the cells are flattened to be in a paving stone-like epithelial cell shape and are arranged into small cell islands, and the bile duct epithelial cells are differentiated mature cells, are easy to age and slow to grow after passage, obviously reduce the proliferation capacity and are difficult to culture. From the origin, the hepatic oval cells have the capacity of bidirectional differentiation, and can be directionally differentiated into hepatic cells and bile duct epithelial cells. Some researchers induce and differentiate the hepatic oval cells into bile duct epithelial cells in vitro; there are also researchers inducing the differentiation of mesenchymal stem cells of bone marrow into hepatocytes or into biliary epithelial cells indirectly or directly.
The human chorion mesenchymal stem cell has the characteristics of mesenchymal stem cells, has multidirectional differentiation potential, can be directionally differentiated into bile duct epithelial cells in vitro through induction, and can be used as a potential source of bile duct bioengineering cells. The following embodiments of the present invention provide a preparation scheme of Human Chorionic Mesenchymal Stem Cells (Human Chorionic mesenchyme Stem Cells, hCMSCs), and establish a foundation for bile duct bioengineering by inducing differentiation into bile duct epithelial Cells in vitro.
Compared with the scheme of inducing and differentiating the hepatic oval cells into the bile duct epithelial cells in vitro and the scheme of 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 of other researchers, the method has the advantages that: the hCMSCs are adult stem cells of mesenchymal sources, are used as seed cells under various conditions, are easy to separate and culture, have wide sources, no additional damage to human bodies and no ethical disputes, have strong proliferation capacity and have stronger stem cell characteristics and multidirectional differentiation potential.
The basic method comprises the following steps:
1) taking placental chorionic villus tissue produced by full-term cesarean section, separating hCMSCs by a homogenate tissue block method, taking 3 rd generation cells to perform flow cytometry detection and identification on hCMSCs phenotypic characteristics, and performing immunofluorescence staining to detect the expression of hCMSCs vimentin and CK-19.
2) In vitro directed differentiation of hCMSCs into hepatic stem cells: inoculating 3 rd generation hCMSCs into a 6-hole plate, adding into an adult liver stem cell culture medium, observing the growth and differentiation forms of cells, collecting cell culture solution after the cells are changed in a circular shape, an oval shape or a polygonal shape, and performing biochemical detection on the contents of alkaline phosphatase, alpha-fetoprotein and albumin.
3) Inducing hepatic stem cells to differentiate into biliary epithelial cells: adding a cholangioblast epithelial cell culture medium into the induced cell 6-pore plate, observing the growth and differentiation conditions of the cells, detecting the expression of cytokeratin CK19 by immunofluorescence staining when the typical morphology of bile duct endothelial cells appears, and identifying the differentiated cells.
Experiments prove that the hCMSCs grow adherently, cells are in long fusiform arrangement after passage, and are arranged in a vortex shape, and flow cytometry identification results show that the hCMSCs express the phenotype of the MSCs, express vimentin and do not express CK-19. After 2 weeks of induction, the cells were observed by inverted differential microscopy to assume a typical oval or polygonal hepatic stem cell morphology, and to assume an atypical nested arrangement. Compared with the method before induction, the hepatic stem cell markers such as alpha fetoprotein and albumin in the cell culture solution after induction are increased compared with the method before induction, the difference has statistical significance (p is less than 0.01), the mesenchymal stem cell marker alkaline phosphatase is reduced, and the difference has statistical significance (p is less than 0.01); after the cells continue to be directionally induced and differentiated for 3 weeks, the cells show typical dendritic bile duct endothelial cell morphological changes, and the induced cell immune expression CK19 is up-regulated.
The specific method comprises the following steps:
hCMSCs in vitro separation and culture
Materials for experiment
The method comprises the steps of obtaining fresh placenta of a full-term fetus of a healthy pregnant woman in a legal way, and controlling the size and the weight of the fresh placenta within a normal range.
hCMSCs isolation and in vitro amplification
1) Selecting placenta of a full-term fetus, mechanically stripping chorion tissues of the placenta, repeatedly washing by PBS (phosphate buffer solution), and shearing the chorion tissues into long blocks of about 1 cm;
2) repeatedly washing with normal saline to remove residual blood, and weighing;
3) homogenizing to 0.1-0.3cm3And washing with physiological saline.
4) Centrifuging at 450-2The incubator for 0.8-1.2 h. Then culture solution is added gently for in vitro culture: adding 2-8ng/ml bFGF human serum-free mesenchymal stem cell culture medium, placing at 35-40 deg.C and 4-6% CO2Culturing in an incubator, changing the culture solution once every 2-4 days, and carrying out passage on the cells according to a conventional method after 12-14 days.
The morphological characteristics of hCMSCs under a microscope are that the growth conditions of primary and passage cells of the hCMSCs are similar, the cell expansion speed is slightly slower than that of P4 before P3 generation, and the cell multiplication speed of each generation is basically stable along with the gradual purification of the cells.
Biological characteristic identification of hCMSCs
1) Culturing cells after P3 generation by using a human serum-free mesenchymal stem cell culture medium, and terminating digestion by using 0.125-0.25% of pancreatin/0.005-0.02% of EDTA;
2) DPBS tuning to 4-6 × 105()40-60 μ l of the seed/stem;
3) respectively adding 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, placing in a refrigerator at 2-8 ℃ for reaction for 15-25min, washing twice with DPBS, and detecting cell phenotype by a flow cytometer.
And (3) detection results: according to the detection result of a flow cytometer, hCMSCs cultured in vitro in P3 generations according to the conventional culture conditions highly express CD73, CD90 and CD105, do not express blood cell markers CD34, CD19, CD45 and HLA-DR, and the phenotype of the hCMSCs is the same as the surface marker proposed by the international placenta-derived stem cell conference.
3h hCMSCs were seeded in six well plates:
1) preparing cell climbing sheet, selecting cell climbing sheet corresponding to six-hole plate, soaking in concentrated sulfuric acid overnight, washing with tap water, soaking in anhydrous alcohol for 5.5-6.5 hr, washing with triple-distilled water for 2-4 times, oven drying in aluminum lunch box, and autoclaving.
2) Cell plating: hCMSCs were cultured to passage 2, counted after 0.1% -0.25% trypsinization and resuspended in culture medium. Before adding cell suspension, small amount of culture medium is dropped into each well according to the size of the slide to adhere the slide and the bottom of the culture plate together, and then the amount of culture medium is 1-3 × 104/cm2And (4) concentration inoculation. Placing at 35-40 deg.C with 4-6% CO2And culturing in a saturated humidity incubator.
Inducing hCSs to differentiate and identify to hepatic stem cells:
after the cell fusion rate in the six-hole plate reaches about 65-85%, washing with PBS for 2-4 times, adding an adult liver stem cell culture medium (human serum-free mesenchymal stem cell culture medium, 0.5-1.5% streptomycin mixed solution, 5-15ng/ml bFGF, 0.5-2% glutamine, 1-10 μ g hepatocyte growth factor), and changing the solution every 3-4 days. Observing the morphological change of the cells by an inverted phase contrast microscope, and biochemically detecting the levels of the alkaline phosphatase, the alpha-fetoprotein and the albumin by taking cell culture fluid after the cells have oval or polygonal changes.
The results show that 3 rd generation hCMSCs are differentiated to the hepatoblasts in vitro, observed by an inverted microscope in the induction culture process, and after 2 weeks of induction, the cells are observed to be gradually arranged in an atypical nest shape from the original vortex shape under a low magnification microscope, and the cells are gradually changed into an oval shape or a polygonal shape from the original spindle shape or long fusiform shape under a high magnification microscope.
Inducing hepatic stem cells to differentiate to bile duct epithelial cells and identifying:
(1) induction of cholangioblast epithelial cells: and (3) when the cells of the six-hole plate are changed into an oval shape or a polygonal shape, abandoning the old culture medium, washing with PBS, adding a cholangiogenic epithelial cell culture medium (a human serum-free mesenchymal stem cell culture medium, 0.5-1.5% streptomycin mixed solution, 0.5-2% glutamine, 0.5-2% hepatocyte growth factor, 1-5% stem cell growth factor and 1-10% epidermal growth factor), changing the solution every 3-4 days, and observing the change of cell morphology under a microscope.
(2) Expression of cellular CK-19 following immunofluorescence induction: and taking out the slide for dyeing when the cells are in a form similar to the form of paving stone-like epithelial cells and have the cell process to change the form of the dendritic bile duct epithelial cells.
Staining cell slide:
a. taking out cell slide, washing with PBS for 2-5 times, each for 2-8 min;
b. dripping 2-6% paraformaldehyde, fixing at 37 deg.C for 25-35min, washing with PBS for 2-5 times, each for 2-8 min;
c. dripping 0.1-0.5% Triton-X100, penetrating membrane at 37 deg.C for 10-20min, washing with PBS for 2-8 times, each for 2-8 min;
e. dripping 10 mu g/ml of immunofluorescent labeled CK-19 antibody to completely cover the specimen, and incubating for 0.5h-1.5h at 37 ℃ in the dark;
f. taking out the slide, washing with PBS for 2-8 times, each time for 2-8min, and staining cell nucleus with DAPI for 15-25 min;
g. taking out the slide, washing the slide for 2-8 times by PBS, and then absorbing the excessive water by filter paper, but keeping the specimen at a certain humidity;
h. the photographs were immediately observed under an inverted fluorescence microscope.
The results show that after the hPMSCs are induced to differentiate into hepatic stem cells, the differentiation of the hepatic 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
Materials for experiment
The method comprises the steps of obtaining fresh placenta of a full-term fetus of a healthy pregnant woman in a legal way, and controlling the size and the weight of the fresh placenta within a normal range.
hCMSCs isolation and in vitro amplification
1) Selecting placenta of a full-term fetus, mechanically stripping chorion tissues of the placenta, repeatedly washing by PBS (phosphate buffer solution), and shearing the chorion tissues into long blocks of about 1 cm;
2) repeatedly washing with normal saline to remove residual blood, and weighing;
3) homogenizing to 0.1-0.3cm3And washing with physiological saline.
4) Centrifuging at 500r/min for 5min, removing supernatant, inoculating the precipitate into several culture dishes according to the weight of inoculating one culture dish with diameter of 100mm to every 5g placenta chorion tissue, and placing the culture dishes upside down in saturated humidity at 37 deg.C and volume fraction of 5% CO2The incubator of (1) for 1 hour. Then culture solution is added gently for in vitro culture: adding 5ng/ml bFGF human serum-free mesenchymal stem cell culture medium, placing at 37 deg.C and 5% CO2Culturing in an incubator, changing the culture solution once every 2-4 days, and carrying out passage on the cells according to a conventional method after 12-14 days.
Morphological characteristics of hCMCs under a microscope are shown in figure 1, the growth conditions of primary and subcultured cells of the hCMCs are similar, the primary cells gradually form flat monolayer cells after being cultured for 2 weeks, the cells grow in a vortex shape, and as the cell density increases, cell bodies become slender and the shape is similar to that of fibroblasts. The primary cells grow slowly, the cells need 3-4 weeks to grow over the bottom of the bottle, the cultured cells can grow and pass more stably, and the proliferation speed of the cells is not obviously slowed down after 10 generations of in vitro culture. 3-7 days after inoculation culture of the primary cells is a growth incubation period, the cells gradually begin to adhere to the wall (figure 1A), and no obvious amplification exists; after 7 days, the cells entered logarithmic growth phase, the cells proliferated actively, the division phase was common, cells with two nuclei appeared, the cell density increased, and they were connected to each other (FIG. 1B). After 8 hours of cell passage, the adherence of the cells can be observed under a phase-contrast microscope, 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 after about 1 week. The cell expansion rate was slightly slower before the P3 generation than after the P4 generation, and the cell doubling rate was substantially constant at each generation as the cells were gradually purified.
A large number of studies at home and abroad show that the mesenchymal stem cells cultured in vitro can be successfully induced and differentiated into hepatocytes, and then form small bile ducts in a sink region; also can induce the in vitro cultured bone marrow mesenchymal stem cells to directly differentiate into bile duct-like epithelial cells.
At present, the mesenchymal stem cells used are mainly of bone marrow origin, which has the advantages of convenience and rapidness, and may have the following defects: the mesenchymal stem cell content in human bone marrow is very low. Each 1 × 104-1×105About 1 mesenchymal stem cell is contained in each mononuclear cell, the number of the obtained primary cells is limited, and the expansion and differentiation capacity and the cell number of the primary cells are obviously reduced along with the age; secondly, the cell acquisition needs invasive operation, so that a donor feels uncomfortable and is not easy to accept, and meanwhile, certain risk exists; and the chance of virus infection is large.
The placental chorion mesenchymal stem cell can replace a bone marrow mesenchymal stem cell, can make up for the defects of the bone marrow mesenchymal stem cell, and has certain superiority. The placenta chorion mesenchymal stem cell has the following advantages: the material is almost not limited. The placenta is a waste product, and can be provided on the basis of the informed consent of healthy lying-in women who give birth normally. The donor has no pain and less pollution chance; the placenta chorion mesenchymal stem cells are more primitive, the immunogenicity is lower, and the number of the obtained primary mesenchymal stem cells is large; and thirdly, no more debate on social, ethical and legal aspects is involved.
Therefore, chorionic mesenchymal stem cells are widely concerned in the field of clinical transformation application. However, the isolation and culture method of the placental chorion mesenchymal stem cells is time-consuming and labor-consuming, the process is complicated and easy to pollute, and the method is an obstacle in using the chorion mesenchymal stem cells.
At present, the separation and culture method of chorionic mesenchymal stem cells mainly comprises a tissue block method and an enzyme digestion method, wherein the enzyme digestion method has high cost, more complicated operation, large pollution opportunity, long enzyme digestion time, easy damage to cells and influence on the activity and passage of the cells. Tissue block adherent culture is generally considered to be superior to tryptic digestion. The traditional tissue block method needs to cut placenta lobule to 1mm3The size of the culture dish is 1cm, and the culture dish is paved in the process, which consumes much time and labor.
The reason for improving and selecting the handheld electric homogenizer tissue block method is that (1) the homogenizer is easy to obtain, the basic function of the homogenizer is used for tissue homogenization, and the instrument is commonly available in a biomedical laboratory. (2) The electric homogenizer is convenient to use, the main body of the device can be moved and disinfected at will, and the rotor of the electric homogenizer can be sterilized. (3) The process is easy to master, the whole process can be seen by naked eyes, and the treatment degree can be adjusted at any time. In conclusion, the improved hCMSCs isolation culture method is an advantage of tissue block adherence culture method combined with the use of a homogenizer. The improved technical system is simple, convenient and quick, improves the yield of the primary hCS MSCs, and lays a foundation for establishing a clinical-grade hC MSCs library.
In addition, in the method, when the chorion is treated by the homogenizer to the size of rice grains, the chorion is washed, most fetal blood can be removed, and when the chorion is treated to the chyle state, the chorion is centrifuged at low speed again, so that fetal blood cells are stored in the supernatant, the influence of erythrocytes on the adherence of hMSCs is effectively removed, 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 possibility of pollution is reduced.
The method improves a separation culture system of the placenta source chorion mesenchymal stem cells on the basis of the traditional tissue block method, and the system has the advantages of simplicity, convenience, rapidness and easiness in obtaining a large amount of primary placenta source chorion mesenchymal stem cells in a short time.
Biological characteristic identification of hCMSCs
1) Culturing cells after P3 generation by using a human serum-free mesenchymal stem cell culture medium, and terminating digestion by using 0.25% pancreatin/0.01% EDTA;
2) DPBS tuning to 5 × 10550 mu l of the seed/grain;
3) respectively adding 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, placing in a refrigerator at 4 ℃ for reaction for 20min, washing twice with DPBS, and detecting cell phenotype by a flow cytometer.
The detection results are shown in fig. 2: according to the detection result of the flow cytometry, the hCMSCs cultured in vitro at the P3 generation according to the conventional culture conditions highly express CD73, CD90 and CD105, do not express blood cell markers CD34, CD19, CD45 and HLA-DR (shown in 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 sheet, selecting cell climbing sheet corresponding to six-hole plate, soaking in concentrated sulfuric acid overnight, washing with tap water, soaking in anhydrous alcohol for 6 hr, washing with triple distilled water for 3 times, drying in aluminum lunch box, and autoclaving.
2) Cell plating: hCMSCs were cultured to passage 2, counted after 0.125% pancreatin digestion and resuspended in culture medium. Before adding cell suspension, small amount of culture medium is dropped into each well according to the size of the slide to adhere the slide and the bottom of the culture plate together, and then the size of the culture plate is 2X 104/cm2And (4) concentration inoculation. Placing at 37 ℃ and 5% CO2And culturing in a saturated humidity incubator.
Inducing hCSs to differentiate and identify to hepatic stem cells:
after the cell fusion rate in the six-hole plate reaches about 70%, washing with PBS for 3 times, adding an adult liver stem cell culture medium (a human serum-free mesenchymal stem cell culture medium, 1% streptomycin mixed solution, b FGF 10ng/ml, 1% glutamine, 5 mu g hepatocyte growth factor), and changing the culture medium every 3-4 days. Observing the morphological change of the cells by an inverted phase contrast microscope, and biochemically detecting the levels of the alkaline phosphatase, the alpha-fetoprotein and the albumin by taking cell culture fluid after the cells have oval or polygonal changes.
The factor combination can induce the bone marrow mesenchymal stem cells cultured in vitro to differentiate into the hepatic stem cells and can promote the proliferation of the hepatic stem cells, and the cells have the dual functions of the bone marrow mesenchymal stem cells and the hepatic stem cells. However, a scheme of adding fetal bovine serum or serum substitute into a basic culture medium such as DMEM is generally adopted, a human serum-free mesenchymal stem cell culture medium is selected, heterologous protein is prevented from being brought in, and a proper amount of factors are added for combination, so that the culture method is more suitable for culturing the human adult stem cells.
As shown in FIG. 4, the 3 rd generation hCMSCs were differentiated into hepatoblasts in vitro, observed by inverted microscope during induction culture, and after 2 weeks of induction, cells were gradually changed from the original vortex-like arrangement to the atypical nested arrangement under low magnification microscope, and cells were gradually changed from the original spindle-like or long fusiform to oval-like or polygonal under high magnification microscope (see FIGS. 4A-B). The results of detection of alkaline phosphatase, alpha-fetoprotein and albumin 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 fluid before and after induced differentiation of hPMSCs to hepatic stem cells
And (4) surface note: compared with the prior induction, P is less than 0.01,
P＜0.01，
P＜0.01
example 5 induction of hepatic stem cells to biliary epithelial cell differentiation and characterization:
induction of cholangioblast epithelial cells: and (3) when the cells of the six-hole plate are changed into an oval shape or a polygonal shape, abandoning the old culture medium, washing with PBS, adding a cholangiogenic epithelial cell culture medium (a human serum-free mesenchymal stem cell culture medium, 1% streptomycin mixed solution, 1% glutamine, 1% hepatocyte growth factor, 2% stem cell growth factor and 5% epidermal growth factor), changing the culture medium every 3-4 days, and observing the morphological change of the cells under a microscope.
The research on in vitro culture of bile duct epithelial cells is less, and not only is the bile duct epithelial cells of adults difficult to separate and purify, but also as differentiated and mature cells, the in vitro culture of the bile duct epithelial cells of adults is easy to age, the proliferation capacity is weakened in the culture process, the in vitro maintenance time is short (the maximum time is 3-4 weeks), and the phenomena of mutation, degeneration, death and the like can occur.
The common culture medium for culturing bile duct epithelial cells comprises DMEM, DMEM/F12, RPMI-1640, BDCM and the like. Different culture media are selected according to the research purpose, and corresponding additional components are added. Growth factors, hormones and amino acids are used as additives to promote proliferation of bile duct epithelial cells. Including keratin cell growth factor, diffusion factor, liver cell growth factor, etc. and has obvious proliferation promoting effect on bile duct epithelial cell.
The method uses hCMSCs to perform two induction methods to form bile duct epithelial cells by improving the formula of a culture medium, the hCMSCs are convenient to obtain, the improved homogenate tissue block method ensures that the hCMSCs are easy to separate and purify, and the hCMSCs are used as undifferentiated mature stem cells, are not easy to age in vitro culture of induced bile duct epithelial cells, have stable proliferation capacity in the culture process, have long in vitro maintenance time, do not age in a short time and other phenomena.
Expression of cellular CK-19 following immunofluorescence induction: and taking out the slide for dyeing when the cells are in a form similar to the form of paving stone-like epithelial cells and have the cell process to change the form of the dendritic bile duct epithelial cells.
Staining cell slide:
a. taking out cell slide, washing with PBS solution for 3 times, each time for 5 min;
b. dripping 4% paraformaldehyde, fixing at 37 deg.C for 30min, and washing with PBS for 5min for 3 times;
c. dripping 0.3% Triton-X100, penetrating membrane at 37 deg.C for 15min, washing with PBS for 3 times, each for 5 min;
e. dripping 10 mu g/ml of immunofluorescent labeled CK-19 antibody to completely cover the specimen, and incubating for 1h at 37 ℃ in the dark;
f. taking out the slide, washing with PBS for 3(2-8) times, each time for 5min, and staining cell nucleus with DAPI for 20 min;
g. taking out the slide, washing the slide for 3 times by PBS, and absorbing the excessive water by using filter paper, but keeping the specimen at a certain humidity;
h. the photographs were immediately observed under an inverted fluorescence microscope.
As shown in fig. 5, after inducing hCMSCs to differentiate into hepatic stem cells, further inducing hepatic stem cells to differentiate into biliary epithelial cells, and after 21 days of induction culture, the cells gradually changed from the original oval shape and polygonal shape to a round-like shape, and appeared to have a cell process, and a stringy phenomenon occurred between the cells, and the cells were arranged to form small "cell islands" in a form similar to the epithelial cell form, and were like a paving stone (see fig. 5A). The expression of CK19 in the cells after induction is detected by immunofluorescence, CK19 positive cells can be seen under an immunofluorescence microscope, and partial cells express strong positive, so that 'pseudopodous' like protrusions can be seen (see figure 5B). Indicating that the cells have the physiological function of bile duct epithelial cells.
The hCMSCs used by the method of the embodiment of the invention are adult stem cells of mesenchymal source, are used as various conditions of seed cells, are easy to separate and culture, have wide source, no additional damage to human bodies and no ethical dispute, have strong proliferation capacity and stronger stem cell characteristics and multidirectional differentiation potential; the stem cells cultured by each placenta chorion can be infused by about 2 ten thousand, so that the industrial preparation is satisfied.
Claims (9)
1. A preparation method of human bile duct epithelial cells is characterized by comprising the following steps:
s1, separating hMSCs by a homogenate tissue block method;
s2, directionally differentiating the hCSs in vitro into hepatic stem cells;
and S3, inducing the hepatic stem cells to differentiate into bile duct epithelial cells.
2. The method for preparing human biliary epithelial cells according to claim 1, wherein the step 1 comprises: selecting placenta of a full-term fetus, mechanically stripping chorion tissues of the placenta, repeatedly washing by PBS, and shearing the chorion tissues into long blocks of 0.8-1.2 cm; repeatedly washing with normal saline to remove residual blood, and weighing; homogenizing to 0.1-0.3cm3Washing with physiological saline; centrifuging at 450-2The incubator for 0.8-1.2 h. Then culture solution is added gently for in vitro culture: adding 2-8ng/ml bFGF human serum-free mesenchymal stem cell culture medium, placing at 35-40 deg.C and 4-6% CO2Culturing in an incubator, changing the culture solution once every 2-4 days, and carrying out passage on the cells according to a conventional method after 12-14 days.
3. The method for preparing human bile duct epithelial cells according to claim 2, further comprising, between steps 1 and 2: hCMSCs were seeded in six-well plates: selecting cell climbing sheets corresponding to a six-hole plate, soaking in concentrated sulfuric acid overnight, washing with tap water, soaking in anhydrous alcohol for 5.5-6.5 hours, washing with triple-distilled water for 2-4 times, drying in an aluminum lunch box, and sterilizing under high pressure for later use; hCMSCs were cultured to passage 2, counted after 0.1% -0.25% trypsinization and resuspended in culture medium. Before adding cell suspension, small amount of culture medium is dropped into each well according to the size of the slide to adhere the slide and the bottom of the culture plate together, and then the amount of culture medium is 1-3 × 104/cm2And (4) concentration inoculation. Placing at 35-40 deg.C with 4-6% CO2And culturing in a saturated humidity incubator.
4. The method for preparing the human bile duct epithelial cells according to claim 1, wherein the step S2 comprises adding the adult liver stem cell culture medium after the cell fusion rate in the hexawell plate reaches 65% -85% and the PBS is washed for 2-4 times, and changing the culture medium every 3-4 days.
5. The method of preparing human biliary epithelial cells according to claim 1, wherein the hepatoblasts culture medium comprises: human serum-free mesenchymal stem cell culture medium, 0.5-1.5% streptomycin mixed solution, 5-15ng/ml of b FGF, 0.5-2% glutamine and 1-10 mu g of hepatocyte growth factor.
6. The method for preparing human bile duct epithelial cells according to claim 5, wherein the step S3 includes: and when the cells of the six-hole plate are changed into an oval shape or a polygonal shape, abandoning the old culture medium, washing with PBS, adding the cholangioblast forming epithelial cell culture medium, changing the medium every 3-4 days, and observing the morphological change of the cells under a microscope.
7. The method of preparing human cholangioblast epithelial cells according to claim 6, wherein the cholangioblast epithelial cell culture medium comprises: human serum-free mesenchymal stem cell culture medium, 0.5-1.5% streptomycin mixed solution, 0.5-2% glutamine, 0.5-2% hepatocyte growth factor, 1-5% stem cell growth factor and 1-10% epidermal growth factor.
8. An adult liver stem cell culture medium comprising: human serum-free mesenchymal stem cell culture medium, 0.5-1.5% streptomycin mixed solution, 5-15ng/ml of b FGF, 0.5-2% glutamine and 1-10 mu g of hepatocyte growth factor.
9. A cholangioblast epithelial cell culture medium, comprising: human serum-free mesenchymal stem cell culture medium, 0.5-1.5% streptomycin mixed solution, 0.5-2% glutamine, 0.5-2% hepatocyte growth factor, 1-5% stem cell growth factor and 1-10% epidermal growth factor.
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| CN115948331A (en) * | 2022-10-17 | 2023-04-11 | 中国人民解放军西部战区总医院 | Method for separating and obtaining mesenchymal stem cells from gall bladder of mouse |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120171168A1 (en) * | 2005-06-17 | 2012-07-05 | Song Sun Uk | Method for treating pancreatitis with mesenchymal stem cells |
| CN104630135A (en) * | 2013-11-06 | 2015-05-20 | 广州熙帝生物科技有限公司 | Method for large scale preparation of liver stem cells, and uses of liver stem cells |
| CN104894064A (en) * | 2015-07-08 | 2015-09-09 | 河南中科干细胞基因工程有限公司 | Culture medium for culturing mesenchymal stem cells |
| CN107142236A (en) * | 2017-04-07 | 2017-09-08 | 广州市妇女儿童医疗中心 | A kind of method that embryo's stones in intrahepatic bile duct epithelial cell is induced to differentiate into the cell model containing ripe bile duct epithelial cell and gallbladder tube structure |
| CN107354130A (en) * | 2017-02-06 | 2017-11-17 | 广州市妇女儿童医疗中心(广州市妇幼保健院、广州市儿童医院、广州市妇婴医院) | A kind of intermembranous mesenchymal stem cells separation method of human placenia |
| CN107523537A (en) * | 2017-10-23 | 2017-12-29 | 洪丰 | A kind of adult people liver derived stem cells system HN and its preparation method and application |
| CN107674857A (en) * | 2017-09-29 | 2018-02-09 | 广州润虹医药科技股份有限公司 | The preparation method and its culture medium group of a kind of epidermal stem cells |
| CN110684722A (en) * | 2019-11-12 | 2020-01-14 | 广东唯泰生物科技有限公司 | Preparation method of mesenchymal stem cells derived from placenta chorion plate tissue |
| CN111304163A (en) * | 2019-11-13 | 2020-06-19 | 上海奈康生物科技有限公司 | Large-scale culture method of umbilical cord mesenchymal stem cells |
-
2020
- 2020-07-20 CN CN202010699889.2A patent/CN111979177B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120171168A1 (en) * | 2005-06-17 | 2012-07-05 | Song Sun Uk | Method for treating pancreatitis with mesenchymal stem cells |
| CN104630135A (en) * | 2013-11-06 | 2015-05-20 | 广州熙帝生物科技有限公司 | Method for large scale preparation of liver stem cells, and uses of liver stem cells |
| CN104894064A (en) * | 2015-07-08 | 2015-09-09 | 河南中科干细胞基因工程有限公司 | Culture medium for culturing mesenchymal stem cells |
| CN107354130A (en) * | 2017-02-06 | 2017-11-17 | 广州市妇女儿童医疗中心(广州市妇幼保健院、广州市儿童医院、广州市妇婴医院) | A kind of intermembranous mesenchymal stem cells separation method of human placenia |
| CN107142236A (en) * | 2017-04-07 | 2017-09-08 | 广州市妇女儿童医疗中心 | A kind of method that embryo's stones in intrahepatic bile duct epithelial cell is induced to differentiate into the cell model containing ripe bile duct epithelial cell and gallbladder tube structure |
| CN107674857A (en) * | 2017-09-29 | 2018-02-09 | 广州润虹医药科技股份有限公司 | The preparation method and its culture medium group of a kind of epidermal stem cells |
| CN107523537A (en) * | 2017-10-23 | 2017-12-29 | 洪丰 | A kind of adult people liver derived stem cells system HN and its preparation method and application |
| CN110684722A (en) * | 2019-11-12 | 2020-01-14 | 广东唯泰生物科技有限公司 | Preparation method of mesenchymal stem cells derived from placenta chorion plate tissue |
| CN111304163A (en) * | 2019-11-13 | 2020-06-19 | 上海奈康生物科技有限公司 | Large-scale culture method of umbilical cord mesenchymal stem cells |
Non-Patent Citations (1)
| Title |
|---|
| 张哲等: "人间充质干细胞的无异源培养体系研究进展" * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115948331A (en) * | 2022-10-17 | 2023-04-11 | 中国人民解放军西部战区总医院 | Method for separating and obtaining mesenchymal stem cells from gall bladder of mouse |
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