CN1995334A - Method for culturing mouse embryo stem cell and its dedicated culture medium - Google Patents
Method for culturing mouse embryo stem cell and its dedicated culture medium Download PDFInfo
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Abstract
The invention discloses a mouse embryo stem cell culturing method and specific cultivating base, which cultures embryo stem cell on the trophoblast cell, wherein the trophoblast cell is epithelial cell or endothelium cell; the cultivating base consists of epithelial cell or endothelium cell and culturing liquid of embryo stem cell, which is high-sugar DMEM with 10-20% embryo cow serum or calf serum with 1. 5-3. 0*10-4mol/L Monothioglycerol, MTG.
Description
Technical field
The present invention relates to a kind of method and special culture media thereof of cultivating mouse embryo stem cell.
Background technology
Embryonic stem cell (embryonic stem cell, ES cell) also claim the ES cell, is a kind of totipotent cell that has that is present in the preceding body early embryo of implantation.Totipotency is the histiocytic ability that differentiation and development becomes three germinal layers.Embryonic stem cell can go down to posterity external the cultivation, keep not breaking up diploid condition with and totipotency, have the ability of formation chimeric animal (chimeric animal).Mouse embryo stem cell is the earliest by Evans and Kaufman (1981) two people and Martin (1981) separation and Culture success and set up clone from the mouse body early embryo respectively.Because it has totipotency, so be widely used in the research of fetal development and embryo engineering aspect.For example in nutrient solution, add different differentiation factors, orientablely induce its differentiation and development cardioblast, lymphocyte and neurocyte, in addition available it turn out organ; Available different allogenic gene transfection embryonic stem cell, or on the embryonic stem cell level, carry out gene knockout or gene targeting, behind in-vitro screening, set up the embryonic stem cell that will have goal gene after the clone have goal gene maybe will be screened and be expelled in host's preimplantation embryos, and be transplanted in the false pregnancy parent uterine cavity and make it to develop into individuality.Thereby set up transgenic animal, gene knockout animal and gene targeting animal, expression and regulation and control and the preparation animal model of human disease etc. of research gene in the differentiation and development process.Because mouse embryo stem cell very easily breaks up in the vitro culture process and loses normal diploid karyotype, and then lose totipotency and lose the ability that forms chimeric animal, so need special culture condition.
The principle of embryonic stem cell vitro culture is: when promoting embryonic stem cell proliferation, keep it and do not break up diploid condition.Embryonic stem cell is in case differentiation promptly loses its totipotency, and the cell that loses the diploid normal karyotype then can reduce the ability that forms mosaic (chimera) greatly, particularly enters kind of an ability that is the formation sexual cell.
The method of extracorporeal culturing embryo stem cell is concluded two big classes at present: feeder layer culture method and no feeder layer culture method are arranged.It is to utilize the various cell preparation conditioned mediums that can secrete the inhibition differentiation factor that no feeder layer is cultivated rule, cultivate mouse embryo stem cell if use such conditioned medium, can not use feeder layer cells, keep when can guarantee mouse embryo stem cell propagation and do not break up diploid condition.Because the non-trophoblast cell culture processes is difficult to keep the mES versatility for a long time in most of laboratories.The main at present nurse cell cultural method that uses.There is the feeder layer culture method mainly to use MEF and the anti-thioguanine of SIM l cell and anti-G-Strophanthin subbreed STO cell as feeder cell; Stop being prepared into the raising individual layer after its division through Mitomycin-C or gamma-radiation processing, embryonic stem cell is planted on such individual layer.MEF and STO can both secrete the factor that promotes embryonic stem cell proliferation and suppress the autonomous differentiation of embryonic stem cell.Suppress the autonomous differentiation factor of embryonic stem cell-LIF content in MEF and the STO culture supernatant at 500～1000IU/ml (secretor type), on MEF and STO matrix, also have LIF (matrix type) simultaneously.The STO cell is to have built anti-thioguanine of SIM l cell and the anti-unabain subbreed (Hogan et al.1994) that is.It can carry out vitro culture for a long time and go down to posterity with frozen.But effect fails to be extensive use of not as MEF.Use MEF as feeder cell cultivate embryonic stem cell be a kind of the earliest and method commonly used.MEF can produce the factor that suppresses the autonomous differentiation of embryonic stem cell and promote embryonic stem cell proliferation, so it can promote embryonic stem cell proliferation effectively and keep it and do not break up diploid condition and totipotency.Usually prepare 12.5～14.5d pregnant mouse and prepare MEF.Because the MEF in the 3rd～5 generation growth and the secretion of the various factor are all vigorous, heteroproteose cell is also less, therefore selects the MEF in the 3rd～5 generation to prepare the trophocyte.When feeder cell are cultivated mES, often in nutrient solution, add the factor-artificial recombination LIF that suppresses the autonomous differentiation of embryonic stem cell.
The basic medium of mouse embryo stem cell is the DMEM of high sugar.Nutrient solution when preparation, also to add beta-mercaptoethanol (multiple compositions such as the LIF of β-ME) or single thioglycerol, glutamine, non-essential amino acid, reorganization and foetal calf serum.The DMEM of high sugar helps adherent, the propagation of inner cell mass of blastaea and the formation of mouse embryo stem cell colony; Foetal calf serum is an indispensable composition in the embryonic stem cell nutrient solution.Promoting to play good effect aspect the embryonic stem cell proliferation; Beta-mercaptoethanol (β-ME) can protect the sulfenyl in desmo enzyme and the protein not oxidized, thus promote the adherent of embryonic stem cell and clone to form; LIF is a kind of multifunctional cytokine of finding sixties Mo.Has the M1 of inducing cell (a kind of myeloid leukemia cell external, myeloid leukaemiccell) to normal differentiation and the autonomous differentiation capability of inhibition embryonic stem cell, and can promote of the growth of the later morula of 8 cell stages to preimplantation embryos, improve the survival rate of blastaea, promote to grow germinal layer cell proliferation and inner cell mass growth.Many tissues and cell all have LIF and LIF receptor expression, grow the expression that germinal layer cell, embryo fibroblast, human bladder cancer cell etc. have LIF as uterine endometrium, decidua, embryo; Embryonic stem cell, M1 myeloid leukemia cell, scavenger cell, embryoma (cancer) cell, adipocyte, neurocyte etc. have the LIF receptor expression.LIF is divided into two types: secretor type (D type) and matrix type (M type).The D type can be secreted in the extracellular fluid, and the M type then anchors on the extracellular matrix.People and mouse LIF gene have 78%～94% homology.There is personnel selection LIF to cultivate mouse embryo stem cell and suppresses the report of its differentiation, but do not see the report of useful mouse LIF cultivator embryonic stem cell.At present, the LIF goods of artificial recombination become commercialized, can be used to prepare the LIF conditioned medium.The LIF optimum dose is 1000IU/ml.When LIF concentration was reduced to 50～100 units/ml, the undifferentiated clone's number of embryonic stem cell can drop to 50%～60%.
Since mES build be since in more than 20 year time, although mouse embryo stem cell vitro culture technology is the most ripe, still there is following shortcoming in above-mentioned culture system: (1) complicated operation: mES vitro culture process comprises that separation and cultivation, MEF trophocyte's preparation, the conditioned medium of preparation, the MEF of 12.5～14.5d pregnant mouse are prepared, the going down to posterity etc. of mES.(2) the MEF lifetime limited, can not go down to posterity for a long time external.Oversize in the subculture in vitro separately time, its ability that produces short multiplicaiton factor and inhibition differentiation factor can weaken even lose.This just needs often to prepare 12.5～14.5d pregnant mouse and prepares MEF.(3) before the plantation embryonic stem cell, if handle MEF with Mitomycin-C, when cleaning was thorough, remaining Mitomycin-C can influence the propagation of embryonic stem cell.(4) in the embryonic stem cell culturing process, the karyomit(e) of dead MEF cell disengages sudden change that may cause embryonic stem cell and the maintenance that influences normal karyotype.(5) nutrient solution complicated component: in the mES culture system, except that basic medium DMEM, foetal calf serum, need to add the beta-mercaptoethanol (LIF of β-ME) or thioglycerin, glutamine, non-essential amino acid, reorganization.Simultaneously, wherein long-time placement such as aminoacid component, reorganization LIF easily causes loss of bioactivity.(6) biomaterial and the reagent that uses pregnant mouse, each seed amino acid, artificial recombination LIF etc. to have very high expense in the traditional cultural method, the national conditions for China's research funding lacks relatively make the research work of relevant mES be subjected to great restriction.
Summary of the invention
The purpose of this invention is to provide a kind of easy, cultivate the method and the special culture media thereof of mouse embryo stem cell effectively, cheaply.
The method of cultivation mouse embryo stem cell provided by the present invention (mES) is on the trophocyte, cultivates mouse embryo stem cell with the embryonic stem cell nutrient solution, and wherein, described trophocyte is epithelium or endotheliocyte.
Described epithelial cell or endotheliocyte can derive from people and Mammals epithelial cell or endotheliocyte.
Described epithelial cell or endotheliocyte specifically can derive from people and mammalian epithelial tissue or endotheliocyte and have the function of spontaneous secretion LIF (comprising spontaneous secretion matrix type LIF and secretor type LIF).
Described epithelial cell and endotheliocyte comprise that building the epithelial cell line, endothelial cell line and former being commissioned to train that are supports epithelial cell and the endotheliocyte that obtains.
Described endotheliocyte is preferably Human umbilical vein endothelial cells, cardiovascular endotheliocyte, myeloid tissue endotheliocyte etc.; Described epithelial cell is preferably tissue-derived epithelial cells such as human bladder epithelium, urogenital tract epithelium, respiratory tract, digestive tube.
Described human bladder cancer's epithelial cell line can be human bladder cancer's epithelial cell T24; Described Human umbilical vein endothelial cells is that Human umbilical vein endothelial cells is ECV-304.
It is isolating from newborn boy baby's umbilical vein that Human umbilical vein endothelial cells is that ECV-304 is derived from Japanese scientist in 1984, have immortal characteristic and do not rely on the endothelial cell line of any cytokine, confirmed afterwards that ECV-304 clone was from human bladder cancer's epithelial cell line T24.
Because of above-mentioned animal epithelial cell and endotheliocyte have similar MEF trophocyte's characteristic as the function that all has spontaneous secretion LIF, for mouse embryo stem cell self, cell fission and propagation provide essential matrix components and necessary cytokine, so can be used as the trophocyte who cultivates mouse embryo stem cell.
In the aforesaid method, described embryonic stem cell nutrient solution can be the DMEM that basic medium is high sugar, adds beta-mercaptoethanol (β-ME) or multiple composition such as thioglycerin and foetal calf serum.
Described nutrient solution preferably in containing the high sugared DMEM of L-glutaminate, adds foetal calf serum or calf serum and thioglycerin, and the quality percentage composition that makes foetal calf serum or calf serum is 10-20%, and the content of thioglycerin is 1.5-3.0 * 10
-4Mol/L.
The present invention selects epithelial cell and endotheliocyte as the trophocyte, is used for the mES cell cultures, has set up a kind of novel mES culture system.This system does not rely on the artificial recombination LIF of external source, does not need to utilize mouse embryo fibroblasts MEF as the trophocyte simultaneously yet.And traditional feeder cell add the factor-artificial recombination LIF that suppresses the autonomous differentiation of embryonic stem cell when cultivating mES in nutrient solution.The present invention utilizes the characteristics of epithelial cell and the spontaneous secretion LIF of endotheliocyte, can avoid increasing the LIF of the artificial recombination of external source.MES culture system provided by the present invention need not pregnant mouse, need not glutamine, need not non-essential amino acid, need not the LIF of artificial recombination.The tradition culture system need possess the equipment and the condition of raising mouse of supporting because of preparation MEF irregularly needs pregnant mouse; Traditional culture system needs multiple amino acids and reorganization LIF simultaneously.The invention enables the cultivation cost of mES to reduce greatly.Utilize method of the present invention, mES can stablize and goes down to posterity more than 20 generations, and can keep undifferentiated diploid condition of mES and versatility, keeps self and increment fast.The method of cultivation mouse embryo stem cell of the present invention, easy and simple to handle, it is simple, with low cost to cultivate composition, has important use and is worth and economic worth.
Description of drawings
Fig. 1 is the trophocyte for the ECV-304 cell, cultivates the mouse embryo stem cell R1 photo of 2 days recovery
Fig. 2 is that human bladder cancer's epithelial cell T24 is the trophocyte, cultivates the mouse embryo stem cell R1 photo of 2 days recovery
Fig. 3 detects mES dryness developed by molecule for RT-PCR
Fig. 4 A be do not handle with Mitomycin-C or the ECV-304 cell of gamma-radiation irradiation as trophoderm, the immunofluorescence of cultivating the Oct3/4 among the mouse embryo stem cell R1 in 20 generations detects photo
Fig. 4 B be do not handle with Mitomycin-C or the ECV-304 cell of gamma-radiation irradiation as trophoderm, the immunofluorescence of cultivating the SSEA-1 among the mouse embryo stem cell R1 in 20 generations detects photo
Fig. 4 C be with Mitomycin-C handle or the ECV-304 cell of gamma-radiation irradiation as trophoderm, the immunofluorescence of cultivating the Oct3/4 among the mouse embryo stem cell R1 in 20 generations detects photo
Fig. 4 D be with Mitomycin-C handle or the ECV-304 cell of gamma-radiation irradiation as trophoderm, the immunofluorescence of cultivating the SSEA-1 among the mouse embryo stem cell R1 in 20 generations detects photo
The mES that Fig. 5 cultivates for the trophocyte for the ECV-304 cell forms 4 days EB after through 20 generations
Fig. 6 A detects the expression of the Neuro D among the EB that forms for Real-time RT-PCR
Fig. 6 B detects the expression of the Sox17 among the EB that forms for Real-time RT-PCR
Fig. 6 C detects the expression of the Brachyury (T) among the EB that forms for Real-time RT-PCR
Embodiment
The method of cultivation mouse embryo stem cell provided by the present invention need not repeated multiple times and prepares pregnant mouse, need not repeated multiple times and prepares MEF, need not repeated multiple times and prepares MEF trophocyte.After the inventive method directly is seeded in the cultivation vessel after handling through gelatin with epithelial cell or endotheliocyte by certain density (with MEF trophocyte's consistent in density of requirement inoculation in the traditional mES culture system), by or handle inoculation mES (require in inoculum density and the traditional mES culture system inoculate mES consistent in density) without mitomycin.After co-cultivation 2-4 days according to 1: the ratio of 2-5 goes down to posterity and continue to cultivate.When going down to posterity, handle, only need according to common attached cell training method trophocyte and the mES cultivation of going down to posterity simultaneously with mitomycin as the epithelium or the endothelium trophocyte of beginning; As beginning epithelium or endothelium trophocyte used mitomycin to handle (treatment process that requires in treatment process and the traditional mES culture system is consistent), before digestion, the collection mES, prepare epithelium or endothelium trophocyte, and the method for digestion and collection mES is with traditional mES cultural method.As use for having built the epithelium that is or endotheliocyte as the trophocyte, can with this epithelium or endothelial cell line carries out frozen and recovery.When epithelium of handling with mitomycin or endothelial cell line and mES do not cultivate altogether, together with epithelium of cultivating altogether or endothelial cell line with mES is frozen simultaneously and recovery.
In order to simplify the research system, with Human umbilical vein endothelial cells be ECV-304 (Japanese DSMZ company, DSMZno.ACC310) or human bladder cancer's epithelial cell line T24 (U.S. ATCC company, ATCC no.HTB-4
TM) be the trophocyte, with mouse embryo stem cell R1 (U.S. ATCC company.ATCC
Number:SCRC-1011) as mES.Below experiment is the research work of carrying out in this system, and its result of study has illustrated the present invention, does not limit the scope of the invention simultaneously (comprise build the epithelium that is or endotheliocyte and former being commissioned to train supported the epithelium that obtains or the mES of endotheliocyte and other types).In addition, method therefor is ordinary method if no special instructions among the following embodiment.Among the following embodiment, except CO
2Percentage composition be outside the volumn concentration, other percentage composition is the quality percentage composition if no special instructions.
Embodiment 1, be that ECV-304 cultivates mouse embryo stem cell as the trophocyte with Human umbilical vein endothelial cells
One, ECV-304 trophocyte's preparation
1, ECV-304 cell cultures
Main experiment material:
Cell: Human umbilical vein endothelial cells is ECV-304 (Japanese DSMZ company, DSMZ no.ACC310), hereinafter to be referred as ECV-304.
Cell culture fluid: (product of Hyclone company, article No. is: add calf serum and thioglycerin (MTG) SH30243.01), making the quality percentage composition of calf serum is 10%, and the content that makes thioglycerin is 1.5 * 10 at the sugared DMEM of height
-4Mol/L.
Digestive system: 0.25% trypsinase adds the solution that 0.02%EDTA obtains with 1: 1 volume ratio.
2, ECV-304 cell recovery:
(1) cell recovery: in frozen liquid nitrogen container, take out freeze-stored cell, insert rapidly in 37 ℃ of-42 ℃ of water-baths, cell is melted fast; Centrifugal removal frozen storing liquid adds the ECV-304 cell culture fluid, puts at 37 ℃, 5% (volume ratio) CO
2, the saturated humidity incubator cultivates.
(2) ECV-304 cell cultures: the cell of recovery changed liquid after 24 hours, continued to cultivate 3-4 days, when treating that the cell growth reaches 70% fusion, by the cultivation of going down to posterity in 1: 3～1: 6.
2, ECV-304 trophocyte's preparation
Main experiment material:
Mitomycin-C: 0.01mol/L, pH that Mitomycin-C 2mg is dissolved in 4ml are among the 7.2-7.5 PBS, are mixed with the mother liquor of 0.5mg/ml; 4 ℃ keep in Dark Place.Before the use, being made into final concentration with above-mentioned cell culture fluid is 10ug/ml.Dilute back 4 ℃ of preservations, be no more than a week.
0.1% aqueous gelatin solution: gelatin 0.1g, ultrapure water 100ml.Autoclaving 45min after the heat of solution a little.4 ℃ of preservations are standby.
(1) prepares: the ECV-304 cell was prepared the trophocyte in 2-3 days in the back of going down to posterity with the ECV-304 trophocyte that Mitomycin-C is handled or gamma-radiation shines.With above-mentioned Digestive system digestion ECV-304 cell, be prepared into cell suspension with above-mentioned cell culture fluid, plant in the culturing bottle of handling with 0.1% aqueous gelatin solution in advance (ware).The cell density of plantation is 1 * 10
4Individual/cm
2With the raising individual layer for preparing at 37 ℃, 5% (volume ratio) CO
2, incubated overnight in the saturated humidity incubator, second day is standby.
(2) prepare with the ECV-304 trophocyte that Mitomycin-C is handled or gamma-radiation shines:
A. when the growth of ECV-304 cell is joined together, add Mitomycin-C in culture supernatant, making final concentration is 10ug/ml, puts back to incubator effect 2～3hr.
Or when the growth of ECV-304 cell was joined together, with the gamma-radiation irradiation, irradiation dose was 3000～10000rad.
B. anticipate culturing bottle (ware) with gelatin.Treatment process is: 0.1% aqueous gelatin solution is sucked in the culturing bottle (ware), get final product can cover culturing bottle (ware) surface, at room temperature leave standstill more than the 2hr.Before the use, inhale and abandon unnecessary aqueous gelatin solution, once with the PBS washing.
C. abandon the feeder cell culture supernatant that contains Mitomycin-C.With PBS washing five times; If handle with gamma-radiation, then save and wash with PBS.
D digests feeder cell.Be prepared into 3 * 10 with above-mentioned cell culture fluid
5The cell suspension of individual/ml density is planted in the culturing bottle of handling with gelatin in advance (ware).The cell count of plantation is: 7.5 * 10
4～1 * 10
5Individual/cm
2
E. at 37 ℃, 5%CO
2, cultivate in the saturated humidity incubator.
F. treat to observe behind the cell attachment,, add the cell of handling, can join together and very close to each other with the assurance cell if find that cell is rare excessively.
The raising individual layer that G. will prepare is placed in the incubator standby.Behind cell attachment, can use in (cell of processing need approximately 6-8 hour can be adherent), will change cell culture fluid before using.ECV-304 trophocyte that Mitomycin-C is handled or gamma-radiation shines and mouse embryo stem cell incubation time altogether are no more than 2 days, must go down to posterity to mouse embryo stem cell, otherwise, cause the differentiation or the apoptosis of mouse embryo stem cell because of ECV-304 trophocyte's death.
Two, built the embryonic stem cell vitro culture that is
1. the cultivation of embryonic stem cell R1 in the ECV-304 trophocyte of not shining with Mitomycin-C processing or gamma-radiation:
(1) the mouse embryo stem cell R1 of recovery is resuspended or will be cultivate 2-4d, eugonic embryonic stem cell R1 and clone and be digested to single cell suspension on traditional mES culture system MEF trophocyte with above-mentioned cell culture fluid, be used to go down to posterity.
(2) (raise monolayer cell density is 1 * 10 to the mouse embryo stem cell R1 direct inoculation of resuspended recovery to the new not ECV-304 raising monolayer cell with Mitomycin-C processing or gamma-radiation irradiation of step 1 preparation
4/ cm
2).(raise monolayer cell density was 1 * 10 to the mouse embryo stem cell R1 that need go down to posterity to the new not ECV-304 raising monolayer cell with Mitomycin-C processing or gamma-radiation irradiation of step 1 preparation with 1: 3～1: 6 ratio transferred species with the ECV-304 cell culture fluid
4/ cm
2).37 ℃, 5% (volume ratio) CO
2, continue in the saturated humidity incubator to cultivate, change liquid every day.The result shows no matter be the mouse embryo stem cell R1 of recovery, the mouse embryo stem cell R1 that still goes down to posterity, and all observing mES after 2 days has embryonic stem cell sample microcolony to occur, and increases in 3-4 days.What Fig. 1 showed is the mouse embryo stem cell R1 photo of cultivating 2 days.Identical with traditional mouse embryo stem cell R1 of 2 days of cultural method cultivation.
(3) cultivate after 2-4 days,, with 1: 3～1: 6 ratio two kinds of cells are changeed simultaneously to be inoculated into in the pretreated cultivation vessel of 0.1% gelatin with the ECV-304 trophocyte and the passage method digestion routinely simultaneously of above-mentioned mES cell of cultivating.This process need not refabrication ECV-304 trophocyte.
(4) continuous passage as stated above.3-4 according to ECV-304 trophocyte's variable density, suitably increases ECV-304 trophocyte after generation, keeps cell density 〉=7.5 * 10
4Individual/cm
2The ECV-304 trophocyte of underpopulation is difficult for keeping the versatility of mES.
(5) frozen and recovery:
Cell cryopreservation: with Digestive system digestion ECV-304 trophocyte and above-mentioned mES cell 5-15 minute, add above-mentioned cell culture fluid termination reaction, supernatant is removed in centrifugal back, uses frozen storing liquid--and-above-mentioned cell culture fluid contains 10-15%DMSO (volume ratio), and re-suspended cell is to 4.0-6.0 * 10
6Individual/ml, each frozen pipe adds the 1ml cell suspension, according to 4 ℃ of placements 0.5-2 hour, places 2 hours for-20 ℃, places 24 hours, and freezes in the liquid nitrogen container for-80 ℃.
The recovery of ECV-304 trophocyte and above-mentioned mES cell is according to the method operation of above-mentioned " ECV-304 cell recovery ".
2. the cultivation of embryonic stem cell in the ECV-304 trophocyte of shining with Mitomycin-C processing or gamma-radiation:
(1) with step 1 preparation with Mitomycin-C handle or the ECV-304 cell inoculation of gamma-radiation irradiation to in the pretreated cultivation vessel of 0.1% gelatin, inoculum density is 7.5 * 10
4Individual/cm
2, as raising individual layer.
(2) second days, that the mouse embryo stem cell R1 of recovery is resuspended or will be cultivate 2-4d, eugonic mouse embryo stem cell R1 clone be digested to unicellular afterwards resuspended with above-mentioned cell culture fluid respectively on traditional mES culture system MEF trophocyte with above-mentioned cell culture fluid.
(3) resuspended mES is inoculated into among the ECV-304 trophocyte that Mitomycin-C is handled or gamma-radiation shines 37 ℃, 5% (volume ratio) CO
2, continue in the saturated humidity incubator to cultivate, change liquid every day.
The result shows no matter be the mouse embryo stem cell R1 of recovery, or the mouse embryo stem cell R1 that cultivates on traditional mES culture system MEF trophocyte, and all observing mES after 2 days has embryonic stem cell sample microcolony to occur, and increases in 3-4 days.Their rate of propagation and embryonic stem cell clone's form is all identical with the mouse embryo stem cell R1 of 2 days recovery of cultivation in not with the ECV-304 trophocyte that Mitomycin-C is handled or gamma-radiation shines that Fig. 1 shows.
(4) cultivate after 2-4 days, continue to go down to posterity by traditional mES cultural method.But need prepare ECV-304 trophocyte in the day before yesterday of going down to posterity:
A. will cultivate 2～3d, eugonic embryonic stem cell clone and be digested to single cell suspension.Digestion method is the same.
B. add above-mentioned cell culture fluid, with 1: 3-1: 6 ratio transferred speciess are to new raising individual layer.
C.37 ℃, 5%CO
2, continue in the saturated humidity incubator to cultivate.
(5) frozen and recovery: with (5) of step 1.
Embodiment 2, be that the trophocyte cultivates mouse embryo stem cell with human bladder cancer's epithelial cell T24
One, T24 trophocyte's preparation
The cell and cell culture fluid in experiment material, other experiment material and experimental technique are all with the step 1 of embodiment 1.
Cell is human bladder cancer's epithelial cell T24 (U.S. ATCC company, ATCC no.HTB-4
TM);
Cell culture fluid is the T24 cell culture fluid: at the sugared DMEM of the height (product of Hyclone company, article No. is: add foetal calf serum and thioglycerin (MTG) SH30243.01), making the quality percentage composition of foetal calf serum is 20%, and the content that makes thioglycerin is 3.0 * 10
-4Mol/L.
Two, built the embryonic stem cell vitro culture that is
Except the trophocyte is that human bladder cancer's epithelial cell T24, cell culture fluid are the T24 cell culture fluid, other experiment material and experimental technique are all with the step 2 of embodiment 1.
The result shows, with handle with Mitomycin-C or human bladder cancer's epithelial cell T24 of gamma-radiation irradiation as the trophocyte, with do not handle with Mitomycin-C or human bladder cancer's epithelial cell T24 of gamma-radiation irradiation as being the trophocyte, the mouse embryo stem cell of cultivating recovery or going down to posterity, all observing mES after 2 days has embryonic stem cell sample microcolony to occur, and increases in 3-4 days.Fig. 2 shows be with Mitomycin-C handle or human bladder cancer's epithelial cell T24 of gamma-radiation irradiation as the trophocyte, cultivate 2 days mouse embryo stem cell R1 photo.Identical with traditional mouse embryo stem cell R1 of 2 days of cultural method cultivation.
Embodiment 3, detect as the mES dryness tagged molecule that the trophocyte cultivates with ECV-304 or human bladder cancer's epithelial cell T24.
One. reverse transcriptase polymerase chain reaction (RT-PCR) detects mES dryness molecule marker at the mRNA horizontal expression:
1. mouse embryo stem cell dryness molecule (tagged molecule): transcription factor Oct3/4, Nanog, Sox2.
2.RT-PCR reaction primer: the synthetic cDNA of reverse transcription uses universal primer OligodT, and pcr amplification said gene primer sequence is respectively: OctP1:5 ' AATGCCGTGAAGTTGGAG3 ', OctP2:5 ' GAAGCGACAGATGGTGGT3 '; NanogP1:5 ' GCCCTGATTCTTCTACCA3 ', NanogP2:5 ' AGATGCGTTCACCAGATAG3 '; Sox2P1:5 ' CCCGTGGTTACCTCTTCC3 ', Sox2P2:5 ' TTCTCCAGTTCGCAGTCC3 '; Internal control gene β-actin P1:5 ' GCTGTCCCTGTATGCCTCT3 ', β-actin P2:5 ' TTGATGTCACGCACGATTT3 '.Wherein, OctP1 and OctP2 are the primers of amplification transcription factor Oct3/4, and NanogP1 and NanogP2 are the primers of amplification transcription factor Nanog, and Sox2P1 and Sox2P2 are the primers of amplification transcription factor Sox2.
3. be collected in the mouse embryo stem cell R1 that cultivated for 20 generations among above-mentioned ECV-304 trophocyte or the human bladder cancer's epithelial cell T24, ordinary method is extracted cell total rna, and carries out the RT-PCR amplification according to ordinary method.The pcr amplification condition is as follows: 94 ℃ of sex change in 4 minutes; (94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 30 seconds) 40 circulations; Last 72 ℃ were extended 10 minutes.Carry out 1.5% agarose gel electrophoresis analysis.
Simultaneously the mouse embryo stem cell R1 that cultivates with the traditional mES culture system that obtains according to following method is in contrast:
(1) handles the MEF feeder cell of having grown in blocks with Mitomycin-C or gamma-radiation, be prepared into the raising individual layer.The preparation method is with embodiment 1.
(2) will cultivate 2～3d, eugonic embryonic stem cell R1 clone and be digested to single cell suspension.Digestion method is with embodiment 1.
(3) add the embryonic stem cell nutrient solution: add among the DMEM 15% calf serum (FBS), 1000IU/ml LIF, 0.1mM β-coloured glaze base ethanol (β-ME) or the single thioglycerol (monothioglycerol) of 0.15mM, 2mM L-glutaminate, 2mM non-essential amino acid, penicillin 100IU/ml, Streptomycin sulphate 100 μ g/ml, with 1: 3～1: 6 ratio transferred species to new raising individual layer.
(4) 37 ℃, 5%CO
2, continue in the saturated humidity incubator to cultivate.
The result shows, in following trophocyte, cultivate among the mouse embryo stem cell R1 in 20 generations, all amplification obtains the Oct3/4 band of 198bp, the Nanog band of 383bp and the Sox2 band of 221bp: a, do not handle with Mitomycin-C or the ECV-304 of gamma-radiation irradiation as the trophocyte; B, with Mitomycin-C handle or the ECV-304 of gamma-radiation irradiation as the trophocyte; C, do not handle with Mitomycin-C or human bladder cancer's epithelial cell T24 of gamma-radiation irradiation as the trophocyte; D, with Mitomycin-C handle or human bladder cancer's epithelial cell T24 of gamma-radiation irradiation as the trophocyte.The mouse embryo stem cell R1 in 20 generations is cultivated in explanation in above-mentioned trophocyte have Oct3/4, Nanog, the expression of Sox2 molecule.Tradition mES culture system still has identical dryness with the mouse embryo stem cell that mES culture system of the present invention is cultivated.
What Fig. 3 showed is not with among the ECV-304 trophocyte that Mitomycin-C is handled or gamma-radiation shines, and cultivates the mouse embryo stem cell R1 in 20 generations.Among Fig. 3, mES: the mouse embryo stem cell R1 that traditional mES culture system is cultivated; Co-mES: be the mouse embryo stem cell R1 that the trophocyte cultivates with the ECV-304 cell that Mitomycin-C is handled or gamma-radiation shines.
Two. cellular immunofluorescence detects the dryness tagged molecule and expresses at protein level:
(1) mouse embryo stem cell dryness molecule: Oct3/4, SSEA-1.
(2) detection antibody: mouse source anti-mouse Oct3/4 monoclonal antibody (R﹠amp; D company, Cat No.MAB1759); Mouse source anti-mouse SSEA-1 monoclonal antibody (R﹠amp; D company, Cat No.MAB2155); The rabbit anti-mouse igg of FITC mark (company of China fir Golden Bridge in Beijing, Cat No.ZF-0304).
(3) be collected in the mouse embryo stem cell of cultivating for 20 generations among the above-mentioned trophocyte, dye mES by the cellular immunofluorescence routine operation, and examine with the DAPI transfect cell.
The result shows, cultivates among the mouse embryo stem cell R1 in 20 generations in following trophocyte, all have the expression of Oct3/4 and SSEA-1 molecule: a, do not handle with Mitomycin-C or the ECV-304 of gamma-radiation irradiation as the trophocyte; B, with Mitomycin-C handle or the ECV-304 of gamma-radiation irradiation as the trophocyte; C, do not handle with Mitomycin-C or human bladder cancer's epithelial cell T24 of gamma-radiation irradiation as the trophocyte; D, with Mitomycin-C handle or human bladder cancer's epithelial cell T24 of gamma-radiation irradiation as the trophocyte.
What Fig. 4 A showed is, handle with Mitomycin-C or the ECV-304 cell of gamma-radiation irradiation as trophoderm, the immunofluorescence of cultivating the Oct3/4 among the mouse embryo stem cell R1 in 20 generations detects photo;
What Fig. 4 B showed is, handle with Mitomycin-C or the ECV-304 cell of gamma-radiation irradiation as trophoderm, the immunofluorescence of cultivating the SSEA-1 among the mouse embryo stem cell R1 in 20 generations detects photo;
What Fig. 4 C showed is, with Mitomycin-C handle or the ECV-304 cell of gamma-radiation irradiation as trophoderm, the immunofluorescence of cultivating the Oct3/4 among the mouse embryo stem cell R1 in 20 generations detects photo;
What Fig. 4 D showed is, with Mitomycin-C handle or the ECV-304 cell of gamma-radiation irradiation as trophoderm, the immunofluorescence of cultivating the SSEA-1 among the mouse embryo stem cell R1 in 20 generations detects photo.
Fig. 4 A and Fig. 4 C are that the cellular immunofluorescence method detects mES dryness tagged molecule Oct3/4: one anti-ly is mouse source anti-mouse Oct3/4 monoclonal antibody, and two anti-ly are the rabbit anti-mouse igg of FITC mark; DAPI is the karyomit(e) dyestuff.Image to left is that the intermediary picture is the nuclei picture of DAPI mark with the fluoroscopic image of the Oct3/4 of FITC mark, and Image to right is the stack of its two images in left side.
Fig. 4 B and Fig. 4 D are that the cellular immunofluorescence method detects mES dryness tagged molecule SSEA-1: one is anti-for being mouse source anti-mouse SSEA-1 monoclonal antibody, and two resist and are the rabbit anti-mouse igg of FITC mark; DAPI is the karyomit(e) dyestuff.Image to left is that the intermediary picture is the nuclei picture of DAPI mark with the fluoroscopic image of the SSEA-1 of FITC mark, and Image to right is the stack of its two images in left side.
Embodiment 4, analyze as the mES versatility that the trophocyte cultivates with ECV-304 or human bladder cancer's epithelial cell T24
One. embryoid body (embry body EB) forms experiment:
1, experiment material:
The EB nutrient solution: basic medium IMDM adds 15% foetal calf serum, 50ng/ml ascorbic acid (vitamin A), 0.5%-1%iron-saturated transferrin (transferrin that iron ion is saturated), 3.0 * 10
-4M MTG, 2mM glutamine, penicillin 100IU/ml, Streptomycin sulphate 100 μ g/ml.Above-mentioned percentage composition is the quality percentage composition.
Low bonding strength φ 6cm culture dish: BRAND company, Cat No.452010.
2, preparation EB.For removal is blended in ECV-304 cell among the mES, to in above-mentioned trophocyte, cultivate the mouse embryo stem cell R1 in 20 generations and be digested to individual cells with Digestive system, PBS washes once, with the DMEM re-suspended cell that contains 10% calf serum, cell transfer in common φ 6cm culture dish, was left standstill 15 minutes in 37 ℃, 5% (volume ratio) CO2, the saturated humidity incubator, carefully draw suspension cell, transfer in another clean φ 6cm culture dish, repetitive operation once.Carefully draw suspension cell (easier to be adherent than mES, after twice sedimentation, removed the ECV-304 cell, the cell of suspension is mES) again because of the ECV-304 cell, centrifugal behind the counting, use EB nutrient solution re-suspended cell instead.According to 1.8-3.0 * 10
4Individual/φ 6cm culture dish, in low bonding strength φ 6cm culture dish, each culture dish adds 6ml EB nutrient solution at least with cell inoculation.The result shows, after suspension culture 3-5 days, the mouse embryo stem cell R1 that in following trophocyte, cultivated for 20 generations all form embryoid body (mEB): a, do not handle with Mitomycin-C or the ECV-304 of gamma-radiation irradiation as the trophocyte; B, with Mitomycin-C handle or the ECV-304 of gamma-radiation irradiation as the trophocyte; C, do not handle with Mitomycin-C or human bladder cancer's epithelial cell T24 of gamma-radiation irradiation as the trophocyte; D, with Mitomycin-C handle or human bladder cancer's epithelial cell T24 of gamma-radiation irradiation as the trophocyte.
That Fig. 5 shows is the EB of mouse embryo stem cell R1 after the EB nutrient solution is cultivated 4 days that cultivated for 20 generations in the ECV-304 trophocyte who handles with Mitomycin-C.
3, Real-time RT-PCR detects three germinal layer tagged molecule that form EB:
(1) three germinal layer tagged molecule: entoderm tagged molecule: Sox17; Mesoderm tagged molecule: Brachyury (T); Ectoderm tagged molecule: Neuro D.
(2) RT-PCR reaction primer: the synthetic cDNA of reverse transcription uses universal primer OligodT, and the primer of pcr amplification Sox17 gene is: Sox17P1:5 ' GCGGACTACAGCAGACAGG 3 ' and Sox17P2:5 ' AGCCACAAATGCCACAAG 3 '; The primer of pcr amplification Brachyury (T) gene is: TP1:5 ' TCCCAGACCTACAGCACC 3 ' and TP2:5 ' CATCCACATTGTTCACCC 3 '; The primer of pcr amplification Neuro D gene is: NeuP1:5 ' CTGCTGAGTCTCGGGATAGTGT 3 ' and NeuP2:5 ' TAGGGTGAAGGCGTTTGG 3 '.The primer of pcr amplification internal control gene β-actin is β-actin P1:5 ' GCTGTCCCTGTATGCCTCT3 ', β-actin P2:5 ' TTGATGTCACGCACGATTT3 '.
(3) collect the EB that cultivated 3-5 days, PBS washes once, with 37 ℃ of digestion of 0.25% pancreatin 10-15 minute, preparation single cell suspension.Extract total RNA.And carry out Real-time RT-PCR according to ordinary method and increase.
The result shows, in following trophocyte, cultivate in the embryoid body that the mouse embryo stem cell R1 in 20 generations forms, all have the expression of the tagged molecule of above-mentioned three germinal layers: a, do not handle with Mitomycin-C or the ECV-304 of gamma-radiation irradiation as the trophocyte; B, with Mitomycin-C handle or the ECV-304 of gamma-radiation irradiation as the trophocyte; C, do not handle with Mitomycin-C or human bladder cancer's epithelial cell T24 of gamma-radiation irradiation as the trophocyte; D, with Mitomycin-C handle or human bladder cancer's epithelial cell T24 of gamma-radiation irradiation as the trophocyte.
That Fig. 6 A shows is the mEB that does not originate with the mouse embryo stem cell R1 that cultivated for 20 generations among the ECV-304 trophocyte that Mitomycin-C is handled or gamma-radiation shines, the express spectra of Neuro D between different differentiation phases;
That Fig. 6 B shows is the mEB that does not originate with the mouse embryo stem cell R1 that cultivated for 20 generations among the ECV-304 trophocyte that Mitomycin-C is handled or gamma-radiation shines, the express spectra of Sox17 between different differentiation phases;
That Fig. 6 C shows is the mEB that does not originate with the mouse embryo stem cell R1 that cultivated for 20 generations among the ECV-304 trophocyte that Mitomycin-C is handled or gamma-radiation shines, the express spectra of Brachyury (T) between different differentiation phases;
Among Fig. 6 A-C, co2d, co3d, co4d, co5d-represent not with Mitomycin-C handle or the ECV-304 trophocyte of gamma-radiation irradiation in cultivate mEB three kinds of expression of gene in 2 days, 3 days, 4 days and 5 days in the mouse embryo stem cell R1 source in 20 generations; Ordinate zou is for detecting the relative expression quantity of gene and internal control gene.
Claims (10)
1, a kind of method of cultivating mouse embryo stem cell is on the trophocyte, cultivates mouse embryo stem cell with the embryonic stem cell nutrient solution, it is characterized in that: described trophocyte is epithelium or endotheliocyte.
2, method according to claim 1 is characterized in that: described epithelial cell derives from the human bladder epithelium; Described endotheliocyte derives from human umbilical vein.
3, method according to claim 2 is characterized in that: described epithelial cell derives from human bladder cancer's epithelial cell line; Described endotheliocyte derives from Human umbilical vein endothelial cells;
Described endotheliocyte comprises builds endotheliocyte and the former foster endotheliocyte that obtains of being commissioned to train that is; Described epithelial cell comprises that building the epithelial cell and former being commissioned to train that are supports the epithelial cell that obtains.
4, method according to claim 3 is characterized in that: described human bladder cancer's epithelial cell line is human bladder cancer's epithelial cell T24; Described Human umbilical vein endothelial cells is that Human umbilical vein endothelial cells is ECV-304.
5, according to arbitrary described method in the claim 1 to 4, it is characterized in that: described embryonic stem cell nutrient solution is in containing the high sugared DMEM of L-glutaminate, add foetal calf serum or calf serum and thioglycerin, the quality percentage composition that makes foetal calf serum or calf serum is 10-20%, and the content of thioglycerin is 1.5-3.0 * 10
-4Mol/L.
6, a kind of substratum that is used to cultivate mouse embryo stem cell is made up of trophocyte and embryonic stem cell nutrient solution; Described trophocyte is epithelium or endotheliocyte.
7, substratum according to claim 6 is characterized in that: described epithelial cell derives from the human bladder epithelium; Described endotheliocyte derives from human umbilical vein.
8, substratum according to claim 7 is characterized in that: described epithelial cell derives from human bladder cancer's epithelial cell line; Described endotheliocyte derives from Human umbilical vein endothelial cells;
Described endotheliocyte comprises builds endotheliocyte and the former foster endotheliocyte that obtains of being commissioned to train that is; Described epithelial cell comprises that building the epithelial cell and former being commissioned to train that are supports the epithelial cell that obtains.
9, substratum according to claim 8 is characterized in that: described human bladder cancer's epithelial cell line is human bladder cancer's epithelial cell T24; Described Human umbilical vein endothelial cells is that Human umbilical vein endothelial cells is ECV-304.
10, substratum according to claim 9, it is characterized in that: described embryonic stem cell nutrient solution is in containing the high sugared DMEM of L-glutaminate, add foetal calf serum or calf serum and thioglycerin, the quality percentage composition that makes foetal calf serum or calf serum is 10-20%, and the content of thioglycerin is 1.5-3.0 * 10
-4Mo1/L.
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| CN101735979B (en) * | 2009-12-31 | 2014-04-30 | 浙江中赢控股集团有限公司 | Method for in vitro amplification of hemopoietic stem cells and precursor cells |
| CN107142237A (en) * | 2011-05-17 | 2017-09-08 | 李晖 | Culture medium, cell culture kit and cell culture processes |
| WO2019209068A1 (en) * | 2018-04-26 | 2019-10-31 | (유)스템메디케어 | Cell line continuously secreting and expressing hla-g proteins, and method for producing same |
| US10696947B2 (en) | 2013-06-11 | 2020-06-30 | Ncardia B.V. | Culture medium composition for maturating cardiomyocytes derived from pluripotent mammalian stem cells |
| CN113355282A (en) * | 2021-07-09 | 2021-09-07 | 首都医科大学附属北京世纪坛医院 | Induction medium and induction method for inducing differentiation of multifunctional stem cells into mesenchymal stem cells from human sources |
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| CN101735979B (en) * | 2009-12-31 | 2014-04-30 | 浙江中赢控股集团有限公司 | Method for in vitro amplification of hemopoietic stem cells and precursor cells |
| CN107142237A (en) * | 2011-05-17 | 2017-09-08 | 李晖 | Culture medium, cell culture kit and cell culture processes |
| US10696947B2 (en) | 2013-06-11 | 2020-06-30 | Ncardia B.V. | Culture medium composition for maturating cardiomyocytes derived from pluripotent mammalian stem cells |
| US12043846B2 (en) | 2013-06-11 | 2024-07-23 | Ncardia B.V. | Cardiomyocytes derived from pluripotent mammalian stem cells |
| WO2019209068A1 (en) * | 2018-04-26 | 2019-10-31 | (유)스템메디케어 | Cell line continuously secreting and expressing hla-g proteins, and method for producing same |
| US11566220B2 (en) | 2018-04-26 | 2023-01-31 | Stemmedicare Co., Ltd. | Cell line consecutively expressing HLA-G protein and method for preparing the same |
| CN113355282A (en) * | 2021-07-09 | 2021-09-07 | 首都医科大学附属北京世纪坛医院 | Induction medium and induction method for inducing differentiation of multifunctional stem cells into mesenchymal stem cells from human sources |
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