CN103031270A - Efficient amplifying and culturing method for biliary epithelial cells - Google Patents
Efficient amplifying and culturing method for biliary epithelial cells Download PDFInfo
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Abstract
The invention discloses an efficient amplifying and culturing method for biliary epithelial cells in rabbit livers and belongs to the technical field of biological medicines. The efficient amplifying and culturing method comprises the following steps of: (1) separating, purifying and culturing the epithelial cells; (2) detecting cell growth lines; and (3) identifying the epithelial cells. The efficient amplifying and culturing method for the biliary epithelial cells, disclosed by the invention, has the advantages as follows: through a multi-step separating method, biliary epithelial cells with higher purities are obtained and a long-time multiplication culturing system for the biliary epithelial cells is established; a fact that the multiplication of the biliary epithelial cells in the livers can be obviously promoted through an Rho kinase inhibitor Y-27632 is first found; and the efficient amplifying and culturing method for the biliary epithelial cells, disclosed by the invention, has the advantages of simpler operation and the like, is suitable for promotion and application and can provide a platform for researches on a plurality of disease mechanisms including biliary atresia, primary biliary cirrhosis and the like.
Description
Technical field
The present invention relates to a kind of efficient amplification and cultural method of rabbit intrahepatic biliary epithelium cell, belong to the biological medicine technology field.
Background technology
The unusual pathology of bile duct epithelial cell can cause the various diseases that comprises Biliary atresia and primary biliary liver cirrhosis etc., but one of them Perfected process of studying these disease incidence mechanism is to set up external bile duct epithelial cell amplification cultivation system.Adopt different separation purification method, researchist priority separation and Culture from people and rodent bile duct tissue goes out bile duct epithelial cell, not yet sees so far the research report of rabbit bile duct epithelial cell separation and Culture.Rabbit is the laboratory animal of commonly using, and it is economical convenient to draw materials, and many-sided physiology of rabbit liver and biochemical indicator have many similar characteristics to human, so is important the replenishing of human liver disease's animal model.
At present, report the epithelial cultural method Main Tissues of normal stones in intrahepatic bile duct piece culture method successfully both at home and abroad, density gradient centrifugation and cell sorting method etc., but these methods exist cell purity not high, in former generation of vitro culture, is a little less than the bile duct epithelial cell multiplication capacity, but can't carry out for a long time amplification cultivation, and research material often needs to relate to whole hepatic tissue, the defectives such as hepatic tissue consumption is larger, as: the cell sorting method is the effective ways that obtain the high purity bile duct epithelial cell, but cell sorting need to have special size separation equipment such as flow cytometer, the individual cells that sorting obtains also and the growth of very suitable bile duct epithelial cell, and the specific surfaces mark that lacks at present bile duct epithelial cell, these factors are restricting the application of carrying out of cell sorting technology; Density gradient centrifugation is the purifying bile duct epithelial cell to a certain extent, but also mixes inevitably cell such as hepatic stellate cell and the fibroblast-like cells of other kinds, thereby cell purity is not high; The tissue mass cell culture advantage is farthest to have kept the epithelial biological characteristics of bile duct in the body, thereby also has a preferably growth activity, but this method can promote the mixed growth of fibroblast-like cells, liver cell and bile duct epithelial cell equally, and purity is lower; Both at home and abroad can be for the normal stones in intrahepatic bile duct epithelial cell strain of using seldom, vicious transformation occurs in Character instability easily; Although the research of more existing bile duct epithelial cell separation and Culture report, but result of study shows existing most of culture system and can't keep for a long time the propagation of cell, even in the situation of adding the cytokines such as EGF and HGF, survival time that yet can only the proper extension cell, effect is very not remarkable.
Have based on this, make the present invention.
Summary of the invention
The efficient amplification and the cultural method that the purpose of this invention is to provide a kind of bile duct epithelial cell.
The technical scheme that realization the object of the invention is taked is as follows:
The epithelial efficient amplification of hepatic duct and cultural method comprise the steps:
(1) epithelial cell separates, and purifying is cultivated;
(2) the Growth of Cells line is measured;
(3) identify.
Wherein:
(1) epithelial cell separates, and purifying is cultivated: get the liver organization of 5-20 gram, be cut into about 1mm
3Fritter, at phosphate buffered saline buffer (phosphate buffer solution, PBS) in behind the washing by soaking 3 times, in 37 ℃ shaking table, DMEM(Dulbecco ' s Modified Eagle Medium with the IV Collagenase Type that contains 5mg/ml) high glucose medium digestion tissue block is 30-60 minute, the centrifugal 5min of collecting cell 1200r/min repeats 3 times and removes collagenase; Purifying hepatic duct epithelial cell, with the hepatic duct epithelial cell behind the purifying respectively with clone's shape density (50 cells/cm
2) be inoculated in the good substratum of configured in advance (DMEM+10%FBS(fetal bovine serum, foetal calf serum)+ITS(Insulin-Transferrin-Selenium), in 37 ℃, 5%(volume volume ratio) and CO
2Middle cultivation is after 6-10 days, and cutting the bile duct epithelial cell colony with the glass minute hand under the aseptic condition is small cell cluster (20-50 cell masses), and is transferred to the cultivation of further going down to posterity in new culture dish or the culture plate.
(2) mensuration of Growth of Cells line: with cell with every hole 5 * 10
3Individual cell is inoculated in the 4 coated orifice plates of matrigel, and every pore volume is 500 μ l, places 37 ℃ 5%(volume ratio) CO
2Cell concn is collected and measured to middle cultivation respectively at the digestion in 5-7 days of cultivating.
(3) evaluation of culturing cell comprises that immunocytochemical stain and bile duct spline structure form, a. immunocytochemical stain: use the 4%(volume ratio) Paraformaldehyde 96 sops up substratum before at room temperature bile duct epithelial cell 20 min(that cultivate of fixing step (1) add Paraformaldehyde 96, and with phosphate buffered saline buffer (phosphate buffer solution, PBS) clean 2-3 time), add the 0.4%(volume ratio) Triton X-100 permeable membrane 15 min, after PBS flushing 3 times; Adding 5%(volume ratio) sheep blood serum, sealing 0.5 h under the room temperature, add primary antibodie, hatching 40 min or 4 ℃ for 37 ℃ spends the night, after removing primary antibodie, PBS carries out DAB(horseradish peroxidase detection kit after washing 3 times) colour developing, detect under the inverted microscope, the cell count that each experiment detects is approximately 1 000;
B. the bile duct spline structure forms: Matrigle is diluted with 1:1 with DMEM, is coated on the thick three-dimensional Matrigle culture system of formation 5mm on culture dish or the culture plate, behind the 1h with the bile duct epithelial cell in 5 generations with 1 * 10
5/ cm
2Cell density is seeded in the upper cultivation of three-dimensional Matrigel 5-7 days, and wherein, nutrient solution is: add 10%FBS(Hyclone company among the DMED/F12), 20ng/ml HGF (hepatocytes growth factor, HGF, pHGF), 1 * ITS and 2mM L-glutamic acid.
Further arranging of technical solution of the present invention is as follows:
In the step (1), described purifying refers to: add the erythrocyte cracked liquid of proper volume (cell pyrolysis liquid with cell volume than being 5:1) in the cell that separates, fully mixing, after the abundant cracking of red corpuscle, trim repeated splitting erythrocyte three times in centrifugal 5 minutes with the speed of 1200r/min; Collecting cell, with cell suspension in DMEM high glucose medium (Dulbecco ' s Modified Eagle Medium), 50g/min, centrifugal 1min repeats three times, collects the unsubstantiality liver cell in the supernatant liquor.
In the step (1), described bile duct epithelial cell medium component is: (alpha Minimum Essential Medium)/the DMEM(mass ratio is 1:1 to α-MEM)+FBS+ITS+10nmol/L Y-27632+100 μ g/ml mycillins.
In the step (1), cultivate culture dish or the culture plate used and all use in advance 250 μ g/ml matrigel in 37 ℃ of coated 1h.
In the separation purification method of technical solution of the present invention, at first adopt the many red corpuscle of erythrocyte cracked liquid cracking quantity clump, then remove mature hepatocytes with density gradient centrifugation and obtain the unsubstantiality liver cell, because mature hepatocytes density and volume are all larger, at 50g/min, can be precipitated to the centrifuge tube bottom in the centrifugal 1min situation, and other cells are retained in the supernatant liquor still in the liver.In the unsubstantiality liver cell of cultivating, the technical program has obtained the cell of bile duct epithelial cell and other type, owing to be the growth of clone's shape density, bile duct epithelial cell is generated large cell colony, we peel off the bile duct epithelial cell colony by microscopically with glass needle on this basis, can obtain highly purified bile duct epithelial cell.
Wherein, matrigel is rich in the extracellular matrixs such as a layer tape albumen, collagen, and this composition is very similar to the interior microenvironment of the body of bile duct epithelial cell; Y-27632 is the inhibitor of Rho signal path, can promote propagation and the survival of multiple epitheliated type stem cell, equally the propagation of bile duct epithelial cell had positive effect, extracellular matrix Matrigel is coated with pre-treatment, promote growth and the propagation of bile duct epithelial cell, realize the long-time amplification cultivation of bile duct epithelial cell, and can impel the cells in vitro continuous passage.
The technical program adopts the matrigel of two dimension as extracellular matrix first, in the situation that Y-27632 exists, but significance ground improves the amplification in vitro efficient of bile duct epithelial cell, and this result shows that Y-27632 may be of universal significance to epithelial proliferation function.
In sum, this research obtains the higher bile duct epithelial cell of purity by the multistep separation method, and set up the long-time multiplication culture system of bile duct epithelial cell, but and find that first Rho kinase inhibitor Y-27632 significance ground promotes the propagation of intrahepatic biliary epithelium cell.The operation of this culture system is comparatively simple, is fit to propagation and employment, and can be research Biliary atresia and primary biliary liver cirrhosis etc. and provide platform in interior various diseases mechanism.
Description of drawings
Fig. 1 be the bile duct epithelial cell cultivated in former generation and going down to posterity and Y-27632 to proliferation (the unsubstantiality liver cell of (A) former culture, white dashed line enclose is bile duct sample epithelial cell; (B) the cultivation epithelial cell that goes down to posterity after the mechanical purity; (C) Y-27632(Y) can significantly promote epithelial propagation (P＜0.05).(A), (B) amplify 100 times);
Fig. 2 bile duct sample epithelial cell immunohistochemical methods is identified ((A) cell expressing CK18; (B) cell expressing CK19; (C) cell expressing GGT; (D) cell expressing vimentin.(A), (B), (C), (D) amplify 100 times);
Fig. 3 bile duct sample epithelial cell bile duct spline structure forms ability, and (culturing cell forms the bile duct spline structure in (A) 3 dimension matrigel systems; (B) immunocytochemistry shows the cell expressing CK7 in the bile duct spline structure, (A) amplifies 100 times, (B) amplifies 200 times).
Embodiment
Materials and methods
1.1 material
1.1.1 10 12 all large new zealand white rabbits of laboratory animal, male and female are not limit, and available from the Shaoxing University Experimental Animal Center, one week of observation is normal rear for testing.
1.1.2 reagent D MEM, α-MEM, ITS, Trypsin-EDTA, the IV Collagenase Type, mycillin and 1 * D-PBS are all available from American I nvitrogen Life Technologies, Inc., hepatocyte growth factor (HGF) is available from Peprotech company (Lian Ke Bioisystech Co., Ltd), the culture plate culture dish is available from NUNC company or Corning company, erythrocyte cracked liquid is available from Tiangen company, foetal calf serum (FBS) is purchased from respectively the Hangzhou folium ilicis chinensis, Hyclone company, Y-27632 is available from Sigma company, and inverted microscope is available from Nikon company.
1.1.3 bile duct epithelial cell substratum: α-MEM/DMEM(1:1)+FBS+ITS+10nmol/L Y-27632+100 μ g/ml mycillins.
Method
1.2.1 the separation of bile duct epithelial cell, purifying and every rabbit of cultivation are got the liver organization of about 5-20 gram, (about 1mm is cut into small pieces
3), in PBS behind the washing by soaking 3 times, in 37 ℃ shaking table, with the DMEM digestion tissue block of the IV Collagenase Type that contains 5mg/ml 30-60 minute, the centrifugal 5min of collecting cell 1200r/min repeated 3 times and removes collagenase, the separation epithelial cell; The erythrocyte cracked liquid that adds proper volume (cell pyrolysis liquid with cell volume than being 5:1) in the epithelial cell that separates, abundant mixing, after the abundant cracking of red corpuscle, put into whizzer, after the trim with the speed of 1200r/min centrifugal 5 minutes, repeat three times, collecting cell, with cell suspension in DMEM solution, 50g/min, centrifugal 1min, repeat three times, collect the unsubstantiality liver cell in the supernatant liquor; Respectively with the unsubstantiality liver cell with clone's shape density (50 cells/cm
2) be inoculated in the good substratum of configured in advance (DMEM+10%FBS(fetal bovine serum, foetal calf serum)+ITS(Insulin-Transferrin-Selenium) in, culture dish or culture plate use 250 μ g/ml matrigel in advance behind 37 ℃ of coated 1h, 37 ℃ 5%(volume volume ratio) CO
2Middle cultivation 6-10 days, after large bile duct epithelial cell sample colony (the plastidogenetic a slice cell that flocks together of homomorphosis) occurring, under the inverted microscope, cutting the bile duct epithelial cell colony with the glass minute hand under the aseptic condition is small cell cluster (20-50 cell masses), and is transferred to the cultivation of further going down to posterity in new culture dish/culture plate.
1.2.2 cell growth curve is measured: with cell with every hole 5 * 10
3Individual cell is inoculated in the 4 coated orifice plates of matrigel, and every pore volume is 500 μ l, places 37 ℃ 5%(volume volume ratio) CO
2Middle cultivation; Respectively at the 5-7 days digestion collecting cells of cultivating, erythrocytometer is measured cell concn.
The evaluation of culturing cell
1.3.1 immunocytochemical stain
With 4% Paraformaldehyde 96 fixed cell 20 min at room temperature, 0.4% (volume ratio) Triton X-100 permeable membrane, 15 min, after PBS flushing 3 times; Adding 5%(volume ratio) sealing 0.5 h under the sheep blood serum room temperature, add primary antibodie, hatching 40 min or 4 ℃ in 37 ℃ spends the night, after removing primary antibodie, PBS flushing 3 times, operation test kit by Dako company carries out the DAB colour developing, detects under the inverted microscope, and the cell count that each experiment detects is approximately 1 000.Not add primary antibodie, two cells that resist detect two anti-non-specific binding situations as negative control group in the DAB operation test kit and add.The primary antibodie source sees Table 1.
Table 1: the antibody that uses in this research
1.3.2 the bile duct spline structure forms experiment Matrigle is diluted with 1:1 with DMEM, is coated on the thick three-dimensional Matrigle culture system of formation 5mm on culture dish/culture plate, behind the 1h with the bile duct epithelial cell in 5 generations with 1 * 10
5/ cm
2Cell density is seeded on the three-dimensional Matrigel culture system and cultivated 5-7 days, and wherein, nutrient solution is: add 10%FBS(Hyclone company among the DMED/F12), 20ng/ml HGF, 1 * ITS and 2mM L-glutamic acid.
Statistical study
Statistic data (3 repetitions) represents that with mean value ± standard error (SE) statistical study adopts SPSS10.0 software to carry out LSD(least-significant difference, the least significant difference) analyze.P＜0.05 is significant difference.
The result
2.1 primary cultured cell form
The unsubstantiality liver cell of centrifugal acquisition is seeded in the culture plate that is coated with matrigel with clone's shape density, and observation of cell is substantially adherent behind the 24h, changes liquid and cleans not adherent cell and a small amount of red corpuscle; Cultivate after 5-7 days, observe the unsubstantiality liver cell of finding former culture under the inverted microscope and two kinds of different cellular fories occur: a kind of cellular form is cube or flat polygon, be " paving stone shape " adherent growth, cell dia is (Figure 1A between 10-15 μ m, cell that white dashed line is enclosed), has very high similarity with bile duct epithelial cell on the form; Another kind is that cellular form is irregular, and some is into fiber-like (Figure 1A).
Cell purification, go down to posterity and long time-histories is cultivated
Under inverted microscope, after with the glass needle that pulls bile duct sample epithelial cell colony being cut into little agglomerate, be transferred to and continue in the new culture dish that is coated with matrigel or the culture plate to cultivate, passage cell shows the polygon epithelial cell form (Figure 1B) of homogeneity, and has no into the growth of other types cell.In containing the culture system of Y-27632, epithelioid cell's colony can pass more than 10 generations continuously, and total survival time was above 3 months; Can only not breed on a small quantity (Fig. 1 C) and add Y-27632 group epithelioid cell, both differences are extremely remarkable.
Epithelioid cell's evaluation
2.3.1 immunocytochemistry dyeing
Epithelioid cell's colony of cultivating for purification Identification is bile duct epithelial cell whether, adopts the Specific marker of multiple bile duct epithelial cell that it is carried out immunocytochemistry and identifies.The result shows: 100% Epithelial granulocyte colony is expressed CK18(Fig. 2 A specifically), CK19(Fig. 2 B), GGT(Fig. 2 C), Vimentin(Fig. 2 D) etc. the Specific marker of bile duct epithelial cell; But do not express hepatocellular mark AFP, the mark Desmin of ALB and hepatic stellate cell and α SMA.This result of study shows: the epithelioid cell of separation and Culture is bile duct epithelial cell.
2.3.2 the bile duct spline structure forms experiment
Confirm that for further the epithelioid cell of separation and Culture is bile duct epithelial cell, we have carried out the bile duct spline structure and have formed experiment.With the cell that covers with 80-90% with 0.05%Trypsin-EDTA digestion after, by 1 * 10
5/ cm
2Cell density is inoculated in the three-dimensional matrigel culture systems, cultivates after 5-7 days, and flat polygonal bile duct epithelial cell forms bile duct spline structure tissue (Fig. 3 A), and expresses CK7(Fig. 3 B).
Stones in intrahepatic bile duct is formed by liver diverticulum head Zhi Fayu, and in the 4th week of people's fetal development, the liver diverticulum head props up epithelial cell and breeds rapidly, form liver cell plate, at the second month of fetal development, form bile canaliculus between the liver cell, the entodermal epithelium cell forms the intrahepatic biliary epithelium cell in succession.The intrahepatic biliary epithelium cell is typical flat cuboidal epithelium form, and cell dia is 10-15 μ m, is the differentiation and maturation cell, and the cell sign thing is GGT, CK7, and CK18, CK19 etc., wherein CK19 is one of bile duct epithelial cell Specific marker.The cell of this research separation and Culture has the typical cell characteristic of bile duct epithelial cell, express the differential protein of bile duct epithelial cell, GGT, CK7, CK18, CK19 etc. form the bile duct spline structure easily in the matrigel of three-dimensional differentiated system, these biological characteristicses have all met the biological characteristics of inside and outside intrahepatic biliary epithelium cell, confirm that the cell of separation and Culture is bile duct epithelial cell.
At first adopt the many red corpuscle of erythrocyte cracked liquid cracking quantity clump in the separation purification method of the present invention, then remove mature hepatocytes with density gradient centrifugation and obtain the unsubstantiality liver cell, because mature hepatocytes density and volume are all larger, at 50g/min, can be precipitated to the centrifuge tube bottom in the centrifugal 1min situation, and other cells are retained in the supernatant liquor still in the liver.In the unsubstantiality liver cell of cultivating, this research has obtained the cell of bile duct epithelial cell and other type, owing to be the growth of clone's shape density, bile duct epithelial cell is generated large cell colony, we adopt microscopically to peel off the bile duct epithelial cell colony with glass needle on this basis, have obtained highly purified bile duct epithelial cell; Mechanically peel need just to have begun certain operation skill, but can skillfully grasp after 2-3 exercise.
Matrigel is rich in a layer tape albumen, the extracellular matrixs such as collagen, and this composition is very similar to the interior microenvironment of the body of bile duct epithelial cell; Y-27632 is the inhibitor of Rho signal path, and the research of recent years finds that Y-27632 can promote propagation and the survival of multiple epitheliated type stem cell, and we infer that Y-27632 has positive effect to the propagation of bile duct epithelial cell equally.This result of study finds that first matrigel with two dimension is as extracellular matrix, but significance ground improves the amplification in vitro efficient (seeing result 2.2) of bile duct epithelial cell in the situation that Y-27632 exists, and this result of study prompting Y-27632 may be of universal significance to epithelial proliferation function.This research finds that also the quality of serum also affects the amplification efficiency of bile duct epithelial cell in addition, in the serum that we have detected at all, and the effect of a certain specific lot number of Hyclone company best (result does not show).
In sum, the technical program obtains the higher bile duct epithelial cell of purity by the multistep separation method, and set up the long-time multiplication culture system of bile duct epithelial cell, but and find that first Rho kinase inhibitor Y-27632 significance ground promotes the propagation of intrahepatic biliary epithelium cell.The operation of this culture system is comparatively simple, is fit to propagation and employment, and can be research Biliary atresia and primary biliary liver cirrhosis etc. and provide platform in interior various diseases mechanism.
Claims (5)
1. efficient amplification and the cultural method of bile duct epithelial cell is characterized in that, comprise the steps:
(1) epithelial cell separates, and purifying is cultivated;
(2) the Growth of Cells line is measured;
(3) identify.
2. efficient amplification and the cultural method of bile duct epithelial cell according to claim 1 is characterized in that, comprise the steps:
(1) epithelial cell separates, and purifying is cultivated: get the liver organization of 5-20 gram, being cut into area is 1mm
3Tissue block, at phosphate buffered saline buffer (phosphate buffer solution, PBS) in behind the washing by soaking 3 times, under 37 ℃ of temperature, digested tissue block 30-60 minute with the DMEM high glucose medium that contains 5mg/ml IV Collagenase Type, collecting cell and centrifugal 5min under 1200r/min, after repeating 3 times, remove collagenase, get the hepatic duct epithelial cell of separation, and carry out purifying; With the hepatic duct epithelial cell behind the purifying respectively with 50 cell/cm
2Be inoculated in the good substratum of configured in advance, in 37 ℃, 5% CO
2Middle cultivation is after 6-10 days, and with the cutting of bile duct epithelial cell colony, per 20-50 cell masses are as one under the aseptic condition, and are transferred to the cultivation of further going down to posterity in new culture dish or the culture plate;
(2) the Growth of Cells line is measured: with cell with every hole 5 * 10
3Individual cell is inoculated in the 4 coated orifice plates of matrigel, places 37 ℃, 5% CO
2Cultivate, respectively at the 5-7 days mensuration cell concns of cultivating, wherein, the every hole of four orifice plates adds the volume 500 μ l of nutrient solution;
(3) evaluation of culturing cell comprises that immunocytochemical stain and bile duct spline structure form,
A. immunocytochemical stain: with the 4% Paraformaldehyde 96 fixing bile duct epithelial cell of cultivating at room temperature, after 20 min minutes, sop up 4% Paraformaldehyde 96, add 0.4%Triton X-100 permeable membrane 15 min, after PBS flushing 3 times; The sheep blood serum of adding 5%, sealing 0.5 h under the room temperature; Add primary antibodie, hatch 40 min or 4 ℃ for 37 ℃ and spend the night, remove primary antibodie, after the PBS flushing 3 times, develop the color and detects, wherein, the cell count of each detection is 1 000;
B. the bile duct spline structure forms: Matrigle is diluted with 1:1 with DMEM, is coated on the thick three-dimensional Matrigle culture system of formation 5mm on culture dish/culture plate, behind the 1h with the bile duct epithelial cell in 5 generations with 1 * 10
5/ cm
2Cell density is seeded on the three-dimensional Matrigel culture system and cultivated 5-7 days, and wherein, nutrient solution is: add 10%FBS among the DMED/F12,20ng/ml HGF, 1 * ITS and 2mM L-glutamic acid.
3. efficient amplification and the cultural method of bile duct epithelial cell according to claim 2, it is characterized in that, described purifying mode is: press cell pyrolysis liquid: cell volume is than being 5:1, in the hepatic duct epithelial cell that separates, add erythrocyte cracked liquid, abundant mixing, after the abundant cracking of red corpuscle, trim, repeated splitting erythrocyte three times in centrifugal 5 minutes with the speed of 1200r/min; Collecting cell, in DMEM solution, the centrifugal 1min of 50g/min repeats three times with cell suspension, collects the unsubstantiality liver cell in the supernatant liquor.
4. efficient amplification and the cultural method of bile duct epithelial cell according to claim 2 is characterized in that: in the step (1), cultivate used culture dish or culture plate and all use in advance 250 μ g/ml matrigel in 37 ℃ of coated 1h.
5. according to claim 1-4 efficient amplification and the cultural method of each described bile duct epithelial cell, it is characterized in that, described bile duct epithelial cell medium component is: α-MEM/DMEM+ FBS+ITS+10nmol/L Y-27632+100 μ g/ml mycillins, wherein, the mass ratio of α-MEM and DMEM is 1:1.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107748130A (en) * | 2017-10-16 | 2018-03-02 | 上海市普陀区中心医院 | A kind of preparation of animal hearts single cell suspension and detection method |
| CN108567995A (en) * | 2017-03-10 | 2018-09-25 | 上海睿泰生物科技股份有限公司 | A kind of preparation method and applications of epithelial cell piece for disease of cornea treatment |
| CN110892061A (en) * | 2017-06-19 | 2020-03-17 | 剑桥企业有限公司 | Method for amplifying bile duct cells |
| CN112342182A (en) * | 2020-10-29 | 2021-02-09 | 浙江大学 | Method for efficiently separating mouse liver bile duct cells |
| WO2023217129A1 (en) * | 2022-05-10 | 2023-11-16 | 上海赛立维生物科技有限公司 | Intrahepatic bile duct precursor-like cell, cell preparation, preparation method, and application |
| CN117233393A (en) * | 2023-11-15 | 2023-12-15 | 四川大学华西医院 | Double-immunohistochemical staining kit and application thereof in identifying benign and malignant bile duct epithelial tumors |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1997732A (en) * | 2004-03-30 | 2007-07-11 | Fac8Cell有限公司 | Culture and use of cells that secrete liver secretory factors |
| CN101864394A (en) * | 2010-06-12 | 2010-10-20 | 昆明亚灵生物科技有限公司 | Method for separating and culturing macaque adult hepatic precursor cells |
| WO2010120785A2 (en) * | 2009-04-13 | 2010-10-21 | The Regents Of The University Of California | Methods and compositions for stem cell cultures |
| CN102257132A (en) * | 2008-11-20 | 2011-11-23 | 森托科尔奥索生物科技公司 | Methods and compositions for cell attachment and cultivation on planar substrates |
| CN102428172A (en) * | 2009-03-20 | 2012-04-25 | 新加坡科技研究局 | Culture of pluripotent and multipotent cells on microcarriers |
-
2013
- 2013-01-05 CN CN201310001672XA patent/CN103031270A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1997732A (en) * | 2004-03-30 | 2007-07-11 | Fac8Cell有限公司 | Culture and use of cells that secrete liver secretory factors |
| CN102257132A (en) * | 2008-11-20 | 2011-11-23 | 森托科尔奥索生物科技公司 | Methods and compositions for cell attachment and cultivation on planar substrates |
| CN102428172A (en) * | 2009-03-20 | 2012-04-25 | 新加坡科技研究局 | Culture of pluripotent and multipotent cells on microcarriers |
| WO2010120785A2 (en) * | 2009-04-13 | 2010-10-21 | The Regents Of The University Of California | Methods and compositions for stem cell cultures |
| CN101864394A (en) * | 2010-06-12 | 2010-10-20 | 昆明亚灵生物科技有限公司 | Method for separating and culturing macaque adult hepatic precursor cells |
Non-Patent Citations (2)
| Title |
|---|
| 刘慧娟 等: "肝脏干细胞的来源与潜能", 《河南科学》 * |
| 张煊 等: "人增生肝外胆管上皮细胞的原代培养及组织学特点", 《中华实验外科杂志》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108567995A (en) * | 2017-03-10 | 2018-09-25 | 上海睿泰生物科技股份有限公司 | A kind of preparation method and applications of epithelial cell piece for disease of cornea treatment |
| CN110892061A (en) * | 2017-06-19 | 2020-03-17 | 剑桥企业有限公司 | Method for amplifying bile duct cells |
| CN107748130A (en) * | 2017-10-16 | 2018-03-02 | 上海市普陀区中心医院 | A kind of preparation of animal hearts single cell suspension and detection method |
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| WO2023217129A1 (en) * | 2022-05-10 | 2023-11-16 | 上海赛立维生物科技有限公司 | Intrahepatic bile duct precursor-like cell, cell preparation, preparation method, and application |
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