CN108251350A - It is a kind of to induce the method broken up to bile duct cell and its special culture media - Google Patents
It is a kind of to induce the method broken up to bile duct cell and its special culture media Download PDFInfo
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- CN108251350A CN108251350A CN201711466727.9A CN201711466727A CN108251350A CN 108251350 A CN108251350 A CN 108251350A CN 201711466727 A CN201711466727 A CN 201711466727A CN 108251350 A CN108251350 A CN 108251350A
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Abstract
The method and its special culture media broken up the invention discloses a kind of induced multi-potent stem cell to bile duct cell.The culture medium is that 2.3% (V/V) fetal calf serum is added on the basis of RPMI1640 culture mediums, 2.7% (V/V) B27,0.2mol/L valines, 1% (V/V) penicillin.The method of inducing differentiation includes the following steps：1) epidermal shape generation protein gene is imported into SW86 cells, obtains the SW86 cells that albumen occurs for high expression activity epidermal shape；2) the SW86 cells of albumen are occurred into for high expression activity epidermal shape and is inoculated with hPSC cells as feeder layer and after carrying out retarded growth processing, it is co-cultured under 37 DEG C, 5%C02 with the special culture media that above-mentioned external evoked pluripotent stem cell differentiation is bile duct cell, obtains bile duct cell.The present invention provides the new ways of an acquisition bile duct cell, have a extensive future.
Description
Technical field
The present invention relates to the methods and its special culture media of Cell differentiation inducing activity, are done carefully more particularly to a kind of induced multi-potent
The method and its special culture media that born of the same parents break up to bile duct cell.
Background technology
By human pluripotent stem cells (hPSC；Including embryonic stem cell hESC and induced multi-potent stem cell hiPSs) systematic function
The ability of property liver cell will provide liver cell source, for drug metabolism study and for handle liver diseases based on cell
Therapy.Liver cell is especially important, because they are responsible for the cell of drug metabolism, and the abnormal shape for being accordingly used in body is raw
The elimination of substance.In view of this effect with individual in terms of the ability of their metabolism certain drug possibility it is different the fact that,
Huge shadow can be had to the drug discovery in pharmaceuticals industry and test by obtaining functional hepatocytes from representative crowd's sample
It rings.New platform in addition to providing drug test, hPSC sources property liver cell can provide potential new treatment for hepatopath.Although
Liver transfer operation provides effective processing for End-stage liver disease, but the shortage of feasible donor organ limits available the method processing
Patient population.Hepatocyte transplantation represents specific for having with the equipment of bioartificial liver developed with hPSC sources property liver cell
The therapy of the redemption life of the patient of the hepatopathy of type.However, these applications are depended on generates ripe metabolic function by hPSC
The ability of cell.Due to the fact that：To control hepatocyte maturation adjusting access understand it is very few, so reproducibility and effectively
It is challenging so far that ground generates this cell.
In view of potential processing and the commercial importance of functional human liver cell, have been directed to optimization and have generated this by hPSC
A little cells have made notable effort.Almost all of method all attempts the critical stage for summarizing the liver development in differentiation culture,
Induction including definitive entoderm, the specificity of the destiny of entoderm to liver, be referred to as liver cell hepatic progenitor cells generation and
Hepatoblast to mature hepatocytes differentiation.It in most of researchs, is induced and broken up with single layer format, wherein known to sequentially adding in
Pathway agonist and antagonist to adjust the early stage of development, induction and liver specialization including entoderm.Pass through this plan
Slightly, it is possible that optimizing the stage of these early differentiations and generating in definitive entoderm, hepatoblast and prematurity liver cell
In highly enriched population, definitive entoderm, hepatoblast and prematurity liver cell are by such as Hex, alpha-fetoprotein and albumin
The expression of label limited.Although these early differentiation stages reasonably establish well, there is no description condition：Such as
Promote the ripe to functional cell of liver cell, functional cell is limited by the activity of stage I and stage II drug metabolic enzyme.
It is very different and in most cases represented not on their maturity state with the population of different schemes generations
Mature hepatocytes.
Invention content
Easy-to-use break up the object of the present invention is to provide a kind of for external evoked multipotential stem cell to bile duct cell
Special culture media.
In order to solve the above technical problems, the present invention takes following technical scheme：For external evoked pluripotent stem cell differentiation
It is that 2.3% (V/V) fetal calf serum is added on the basis of RPMI1640 culture mediums for the special culture media of bile duct cell,
2.7% (V/V) B27,0.2mol/L valines, 1% (V/V) penicillin.
Second object of the present invention is to provide the side that a kind of external evoked multipotential stem cell directed differentiation is bile duct cell
Method.
External evoked pluripotent stem cell differentiation provided by the present invention is the method for bile duct cell, is included the following steps：
1) albumen (epimorphin, EPM) channel genes are occurred into for epidermal shape into SW86 cells, obtains high expression activity
The SW86 cells of albumen occur for epidermal shape；
2) the SW86 cells that high expression activity epidermal shape to that albumen occurs as feeder layer and carry out retarded growth processing
Afterwards be inoculated with hPSC cells, with the special culture media that above-mentioned external evoked pluripotent stem cell differentiation is bile duct cell 37 DEG C, 5%
CO2Lower co-cultivation promotes hPSC cells to break up to bile duct cell and forms tube-like structures, obtains bile duct cell.
Can protein gene transfection SW86 cells conventionally be occurred into for epidermal shape, such as be sent out by containing epidermal shape
The recombinant eukaryon expression vector of raw albumen gene utilizes slow virus carrier system or utilizes lipofection etc. by epidermis shape
State occurs protein gene and imports SW86 cells.
High expression activity epidermal shape occurs in step 2) method that the SW86 cells of albumen carry out retarded growth processing
For：The mitomycin of a concentration of 15-20 μ g/ml of SW86 cells of albumen occurs for the high expression activity epidermal shape after will be adherent
In 37 DEG C, 5%CO2Lower processing cell 1h.
The bile duct cell induced in aforementioned manners also belongs to protection scope of the present invention.
The method and its special culture media broken up the present invention provides a kind of induced multi-potent stem cell to bile duct cell.The party
It by the mode hPSC cell differentiations of co-cultivation is bile duct cell that method, which is, and the technical solution of use is as follows：Pass through liposome
Infection protocol is by EPM channel genes SW86 cells, then passes through limiting dilution method and expression is stablized in the unicellular inocalation method screening of streaming
The SW86 cells of EPM are simultaneously identified, then T7-EPM-SW86 cells as feeder layer and are carried out retarded growth processing and are followed by
Kind of hPSC cells, make hPSC cultivate in vitro under conditions of Self-differentiation for bile duct cell and form tube-like structures.Morphology
It observes, the verification result of gene level shows to be obtained expression with the method and culture medium of the present invention and be had Within Human Biliary Tract correlating markings
The bile duct cell of Yp, Cx43, Aquaporin-1 etc..The present invention provides the new way of an acquisition bile duct cell, application prospects
It is wide.
Description of the drawings
Fig. 1 is the detection of bile duct marker gene expression when hPSC cells are co-cultured with T7-EPM-SW86 cells
Specific embodiment
Example below is used herein to demonstration the preferred embodiments of the invention.Those skilled in the art, it will be appreciated that under
State the technology disclosed in example represent inventor discovery can be used for implement the present invention technology, therefore can be considered as implementation this
The preferred embodiment of invention.But those skilled in the art should be understood that specific reality disclosed herein according to this specification
Many modifications can be made by applying example, still can be obtained identical or similar as a result, rather than away from the spirit or scope of the present invention.
Unless otherwise defined, the technology in the term and fields of the present invention of all technologies as used herein and science
Personnel institute is normally understood equivalent in meaning, and being disclosed reference and their materials of reference will all be incorporated.
Those skilled in the art will recognize or just will appreciate that by routine test many described here
Invention particular embodiment many equivalent technologies.These will be equally comprised in claims.
Embodiment 1, external evoked pluripotent stem cell differentiation are bile duct cell
The external evoked pluripotent stem cell differentiation of method with the present invention is bile duct cell, is included the following steps：
First, stablize the screening of the SW86 cell monoclonals of expression EPM
By the plasmid T7-EPM-pQXCIN of carrying EPM genes, (construction method is shown in：Hongbin Z, Elaine FR,
Claude S, et al.The Epimorphin Gene Is Highly Conserved among Humans, Mice, and
Rats and Maps to Human Chromosome 7, Mouse Chromosome 5, and Rat
Chromosome12.Genomics, 1996,37：386-389.) after amplification, sequencing result are correct, lipofection is utilized
Plasmid is imported in SW86 cells (this laboratory cell library preserves cell line), is as follows：
1) under the conditions of 37 DEG C, 5%CO2, culture SW86 cells (when cell density is high, can obtain most to 90-95% is covered with
Good result).Replacing within 5 hours before transfection complete medium, [(Hyclone is public for HD-DMEM (GIBCO companies)+10% fetal calf serum
Department)+0.2mol/L valines (sigma companies)].
2) T7-EPM-pQXCIN Plasmid DNA is diluted to a concentration of 200 μ with best medium OPTI-MEM (GIBCO companies)
g/ml。
3) with OPTI-MEM dilution liposome Lipofectamine 2000 (Invitrogen companies) to a concentration of 400 μ
G/ml, (liposome Lipofectamine 2000 must be combined in 30 minutes with DNA, otherwise activity drop within 5 minutes for incubation at room temperature
It is low).
4) diluted T7-EPM-pQXCIN Plasmid DNA with diluted Lipofectamine 2000 is mixed, be incubated at room temperature
20 minutes to form DNA- liposomes Lipofectamine 2000 compound (compound ambient-temp-stables 6 hours；Mixture can be in
Cloud, but transfection is not interfered).
5) culture solution is sucked out, cell is washed 2 times with serum-free medium (GIBCO companies), serum-free nonreactive is added in every ware
Raw element culture solution, adds 2000 compounds of DNA- liposomes Lipofectamine, 5 is cultivated under the conditions of 37 DEG C, 5%CO2
Hour, complete medium is replaced, continues to cultivate, is passed in due course.
6) it after transfectional cell is passed on by 10: 1, cultivates 24 hours, changes into containing G418's (final concentration 400-600 μ g/mL)
HD-DMEM culture solutions (GIBCO companies) are screened, and replacement culture medium is primary every three days, treat that cellular control unit is all dead,
When macroscopic cell clone occurs in its orifice plate, start picking cell clone (alternative：Transfectional cell is thin using streaming
The inoculation of born of the same parents' instrument is unicellular).
Then, the mRNA and albumen of transfectional cell (using the SW86 of untransfected as control) is extracted, carries out RT-PCR inspections respectively
It surveys, using β-actin as internal reference, empirical tests EPM genes on gene and protein level express by height, illustrates that EPM genes have become
It rotates into work(in SW86 cells, obtains the SW86 cells of high expression activity EPM genes, be named as T7-EPM-SW86.
2nd, hPSC cells are induced to differentiate into bile duct cell and form tube-like structures
Differential medium：3% (V/V, 3-5%) fetal calf serum is added on the basis of RPMI1640 culture mediums
(fetal calfserum, FCS), 2% (V/V, 1-3%) B27 and 1% (V/V, 0.5-1.5%) penicillin.
1) routine culture EPM-SW86 cells, 0.25% pancreatin digestion, are counted, are inoculated in 6 orifice plates with 3 × 104/hole
Culture；
2) after EPM-SW86 cells to be seeded are adherent, with the mitomycin processing cell 1h of a concentration of 15-20 μ g/ml
(37 DEG C, 5%CO2, during which cell is shaken up every 15min) and with retarded growth, 1 × PBS washs 4-5 time, and to remove, to remain mitogen mould
Element can be inoculated with；
3) the hPSC cells normally cultivated are taken, with 0.25% trypsin digestion, 1,200rpm centrifugation 5 minutes counts, with
1.5-3 × 105/hole is inoculated in 6 orifice plates in step 2, per 4 hole of plate, wherein 2 holes are the negative control (hPSC not co-cultured
Cell), in 37 DEG C, 5%CO2, co-culture under the conditions of saturated humidity.
12 h after inoculation starts to observe, and observation in every 24 hours later is primary.The results show that compared with negative control,
HPSC cells and T7-EPM-SW86 (sep) cell form tube-like structures after co-cultivation, cellular contraction and edge in structure
The stretching of tube-like structures direction.
3rd, the verification that hPSC cells bile duct breaks up in co-culture system
HPSC cell of the step 2 through co-cultivation is taken, extraction mRNA carries out RT-PCR, and primer is designs according to gene order
Custom primer.The results are shown in Figure 1, compared with negative control cell, the table of bile duct mark Yp, Cx43, Aquaporin-1 etc.
It is raised up to amount, shows hPSC cells through inducing differentiation into as bile duct cell.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To be made various changes or modifications to the present invention, such equivalent forms equally fall within the model that the application the appended claims are limited
It encloses.
Claims (5)
1. it is on the basis of RPMI1640 culture mediums for the culture medium that external evoked pluripotent stem cell differentiation is bile duct cell
It is added to 2.3% (V/V) fetal calf serum, 2.7% (V/V) B27,0.2mol/L valines, 1% (V/V) penicillin.
2. a kind of external evoked pluripotent stem cell differentiation is the method for bile duct cell, include the following steps：
1) epidermal shape generation protein gene is imported into SW86 cells, obtains high expression activity epidermal shape and albumen occurs
SW86 cells；
2) using high expression activity epidermal shape occur albumen SW86 cells as feeder layer and carry out retarded growth handle be followed by
Kind of hPSC cells are the special culture media of bile duct cell 37 with external evoked pluripotent stem cell differentiation described in claim 1
DEG C, 5%CO2Lower co-cultivation, obtains bile duct cell.
3. abductive approach according to claim 2, it is characterised in that：By containing epidermal shape in the step 1)
The recombinant eukaryon expression vector of protein gene is sent out epidermal shape using slow virus carrier system or using lipofection
Raw albumen channel genes SW86 cells.
4. abductive approach according to claim 2, it is characterised in that：To high expression activity epidermal shape in the step 2)
Occur albumen SW86 cells carry out retarded growth processing method be：Egg occurs for the high expression activity epidermal shape after will be adherent
White SW86 cells are with the mitomycin of a concentration of 15-20 μ g/ml in 37 DEG C, 5%CO2Lower processing cell 1h.
5. the bile duct cell induced with any one of claim 2-4 the methods.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111304147A (en) * | 2018-12-11 | 2020-06-19 | 中国科学院分子细胞科学卓越创新中心 | Method for preparing functional bile duct cells in large scale by using endoderm stem cells and application thereof |
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| CN101418285A (en) * | 2008-11-28 | 2009-04-29 | 中国人民解放军军事医学科学院野战输血研究所 | Method for inducing hepatic oval cells differentiation to bile duct cells and its special culture medium |
| CN101497872A (en) * | 2008-02-02 | 2009-08-05 | 中国人民解放军军事医学科学院野战输血研究所 | Method for inducing human embryo stem cell for directional differentiation into hepatocyte and special culture medium |
| CN107299080A (en) * | 2017-05-24 | 2017-10-27 | 清华大学 | By the embryo stem cell external evoked method and its used medium for ovarian follicle in people source |
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2017
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101497872A (en) * | 2008-02-02 | 2009-08-05 | 中国人民解放军军事医学科学院野战输血研究所 | Method for inducing human embryo stem cell for directional differentiation into hepatocyte and special culture medium |
| CN101418285A (en) * | 2008-11-28 | 2009-04-29 | 中国人民解放军军事医学科学院野战输血研究所 | Method for inducing hepatic oval cells differentiation to bile duct cells and its special culture medium |
| CN107299080A (en) * | 2017-05-24 | 2017-10-27 | 清华大学 | By the embryo stem cell external evoked method and its used medium for ovarian follicle in people source |
Non-Patent Citations (1)
| Title |
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| JIA Y等: "epimorphin in bile duct formation of rat liver epithelial stem-like cells: Involvement of small G protein RhoA and C/EBPβ", 《JOURNAL OF CELLULAR PHYSIOLOGY》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111304147A (en) * | 2018-12-11 | 2020-06-19 | 中国科学院分子细胞科学卓越创新中心 | Method for preparing functional bile duct cells in large scale by using endoderm stem cells and application thereof |
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Application publication date: 20180706 |