CN1298843C - Human embryonic stem cells culture medium without dependent feeding cell - Google Patents
Human embryonic stem cells culture medium without dependent feeding cell Download PDFInfo
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- CN1298843C CN1298843C CNB2005100113250A CN200510011325A CN1298843C CN 1298843 C CN1298843 C CN 1298843C CN B2005100113250 A CNB2005100113250 A CN B2005100113250A CN 200510011325 A CN200510011325 A CN 200510011325A CN 1298843 C CN1298843 C CN 1298843C
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Abstract
The present invention relates to a human embryo stem cell culture medium (LD-1 cell culture medium) independent of feeder cells, particularly to a human embryo stem cell culture medium independent of mouse embryo fibroblasts or any other cells, which is obtained through adding a few growth factors supporting the self renewal of human embryo stem cells to an embryo stem cell culture medium of an ordinary person, wherein the growth factors can be fiber growth factors 2(bFGF), Noggin, etc. After the factors are added to the embryo stem cell culture medium of an ordinary person, the embryo stem cell culture medium of an ordinary person can be directly used for culturing human embryo stem cells, and any components from mouse embryo fibroblasts or any other cells do not need to be replenished.
Description
Technical field:
The present invention relates to a kind of people's embryonic stem cells substratum (LD1 cell culture medium) that does not rely on feeder cell, be specifically related in the general population's embryonic stem cells substratum, add the factor of some backer's embryonic stem cells selfs, and do not rely on people's embryonic stem cells substratum of mouse embryo desmocyte or other any cells.
Background technology:
Professor Thomson of Univ Wisconsin-Madison USA was first successfully with the most original undifferentiated inner cell mass (Inner Cell Mass among the early stage people embryo (after fertilization 5-7 days blastaeas) in 1998, ICM) separate, be inoculated into (mouse embryonic fibroblasts on the mouse embryo desmocyte that is monolayer culture, MEFs), continue vitro culture, go down to posterity, and keep its undifferentiated state, and be referred to as human embryo stem cell (hereinafter to be referred as for people's embryonic stem cells or hES cell) (Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, MarshallVS, Jones JM.Embryonic stem cell lines derived from human blastocysts.Science.1998 Nov 6; 282 (5391): 1145-7.).According to fetology research, all tissue, the organs of human body all are differentiated to form by these ICM, and ICM is considered to have totipotency (Totipotency).Therefore, the hES cell brings 21 century medical science revolution probably.
The purpose of hES cell research finally is to be used for the clinical treatment patient, but existing in the world at present hES clone in the process of separating and cultivating, all needs mouse embryo desmocyte as sustenticular cell, thereby may contain certain unknown objectionable constituent of animal, as animal virus etc.Such hES cell obviously is unsuitable for clinical application, because the hES cell that will so cultivate is transplanted in patient's body, just might bring the mankind certain unknown animal virus or other objectionable impurities.
In addition, the preparation of mouse embryo desmocyte (MEFs) is not only consuming time, cost is high and be subjected to the influence of a plurality of link labile factors, and the hES cell cultures based component that contains the MEFs composition is all non-constant.This brings a lot of difficulties and inconvenience for the research of hES cell experiment, and is for example repeated bad etc.
Therefore, setting up the hES clone that does not rely on mouse embryo desmocyte is the key subjects that this area need solve.
Be head it off, researchists attempt to avoid the hES cell inoculation is cultivated to the method on the mouse embryo desmocyte, as human conditioned mediums such as calendar year 2001 Xu (promptly on MEFs, spend the night and obtained the common hES substratum of its efficiency factor), cultivation is seeded in the hES cell on the Matrigel, (Xu C succeeds, Inokuma MS, Denham J, Golds K, Kundu P, Gold JD, Carpenter MK.Feeder-free growth of undifferentiatedhuman embryonic stem cells.Nat Biotechnol.2001 Oct; 19 (10): 971-4.).Like this, be actually the hES cell is become indirect contact with the relation of MEFs by directly contacting, really be not completely free of dependence MEFs.
According to research, MEFs not only helps the growth of hES cell attachment, the more important thing is that MEFs contains unknown material of certain (a bit), has supported the self of hES cell.People infer, may secrete certain (a bit) nutritional factor or meta-bolites in the mouse embryo desmocyte and make hES clone carry out self.In theory, are any material if can make these nutritional factor clear, perhaps find nutritional factor with equal biological effect, its () to be added in the common hES cell culture medium, the dependence to MEFs just can be thoroughly broken away from the cultivation of hES cell.But do not see the relevant report of this type of work success at present.
Summary of the invention:
The objective of the invention is to find not rely on mouse embryo desmocyte (MEFs), also do not add any other feeder cell composition, and can keep the material that people's embryonic stem cells (hES) cell carries out self;
Another object of the present invention provides a kind of novel people's embryonic stem cells substratum that does not rely on mouse embryo desmocyte or other any cellular constituents.
The present invention has found the somatomedin of some support hES cell growth effects, after adding in the common hES cell culture medium according to certain concentration these factors, can make it be directly used in the cultivator embryonic stem cells, and no longer need to replenish any composition from mouse embryo desmocyte or other any cells.
People's embryonic stem cells substratum with the somatomedin of the present invention's discovery is prepared does not rely on mouse embryo desmocyte or other any cells, has reached the object of the invention.
The factor of the support hES cell growth that the present invention finds is bFGF (fibroblast growth factor 2) and Noggin (as available Recombinant Mouse Noggin/Fc Chimera etc.).
People's embryonic stem cells substratum provided by the invention (LD-1 cell culture medium) is bFGF and the Noggin that adds extraordinary concentration in common hES cell culture medium.
In people's embryonic stem cells substratum provided by the invention, the working concentration of bFGF and Noggin is very important.The inventor has just found suitable bFGF and Noggin working concentration through repeatedly exploring, i.e. bFGF:4-50ng/ml substratum, Noggin:100-1000ng/ml substratum; The too small then effect of concentration is not good, and the excessive then cost of concentration is too expensive.
More preferred concentration is bFGF:10-40ng/ml substratum, Noggin:250-500ng/ml substratum.
Described common hES cell culture medium is the various hES cell culture mediums that those skilled in the art use always, such as, available common hES cell culture medium as the prescription among the embodiment one.
Experimental results show that, in common hES cell culture medium, add bFGF (10-40ng/ml) and Noggin (250-500ng/ml) simultaneously, the hES cell can not only normal growth, can also repeatedly go down to posterity, and keep its undifferentiated state and be divided into interior, in, the potential of outer three germinal layer cells.
In the above-mentioned people's embryonic stem cells substratum that provides of the present invention, also can add Bovine Fibronectin (a kind of fibronectin is called for short Fibronectin).Experiment showed, the cloning efficiency that can increase the hES cell, and accelerate its speed of growth.The consumption of Fibronectin can be 0.5-50 μ g/ml), preferable amount is 0.5-5 μ g/ml.
Perhaps, in people's embryonic stem cells substratum of above-mentioned adding bFGF of the present invention and Noggin, add heparin (Heparin).Experiment showed, the consumption that can reduce the more expensive bFGF of price, reduce by half to major general bFGF consumption.Can reduce cost like this.
The consumption of heparin can be 5-50 μ g/ml), preferable amount is 10-30 μ g/ml.
Also can in the above-mentioned people's embryonic stem cells substratum that provides of the present invention, add Fibronectin and Heparin simultaneously.
The above-described bFGF of the present invention, Noggin, Fibronectin and four kinds of materials of Heparin all can be bought by mode such as commercially available.
Embodiment:
Agents useful for same of the present invention and experiment material are as described below:
1, cell
A) hES cell (H1): provide by WiCell institute of Univ Wisconsin-Madison USA;
B) MEFs after the radiation treatment (mouse embryo desmocyte): the intravital mouse embryo of the pregnant mouse of ICR that is provided by Peking University's Experimental Animal Center is prepared from; (method is referring to Thomson JA, Itskovit (z-Eldor J, ShapiroSS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM.Embryonic stem celllines derived from human blastocysts.Scienc.1998 Nov6; 282 (5391): 1145-7.)
2, cell culture medium
A) common hES cell based
Prescription one:
D-MEM/F12(Invitrogen，#11330-032) 200ml
KnockOutTM Serum Replacement(Invitrogen，#10828-028) 50ml
NEAA (non-essential amino acid, Invitrogen, #11140-050) 2.5ml
Glutamine/mercaptoethanol solution (seeing prescription three) 1.25ml
BFGF solution (seeing prescription four) 0.5ml
B) mouse embryo desmocyte substratum
Prescription two:
D-MEM(Invitrogen，#11965-092) 225ml
FBS(Fetal Bovine Serum，Invitrogen，#16000-044) 25ml
NEAA (non-essential amino acid, Invitrogen, #11140-050) 2.5ml
200mM glutamine+mercaptoethanol solution
*1.25ml
Prescription three:
200mM L-Glutamine(Invitrogen，#21030-081) 10ml
2-Mercaptoethanol(Sigma，M-7522) 7μl
Prescription four:
rh bFGF(BD，#354060) 10μg
0.1％BSA in PBS 5ml
3, somatomedin:
a)bFGF(BD，#354060)
b)Noggin(R&D，719-NG-050)
c)Recombinant Mouse Wnt3a(R&D 1324-WN-002)
d)Fibronectin(BD，#356008)
4、PBS(Phosphate Buffered Saline，Invitrogen，#14190-250)
5、0.2％Dispase in D-MEM/F12
Prescription five:
Dispase(Invitrogen，#17105-041) 100μg
D-MEM/F12(Invitrogen，#11330-032) 50ml
6、Matrigel(BD，#354234)
7, antibody
a)Oct-4(Chemicon MAB4305，mouse IgG1)
b)SSEA-4(Chemicon MAB4304，mouse IgG3)
c)TRA-1-60(Chemicon MAB4360，mouse IgM)
d)TRA-1-81(Chemicon MAB4381，mouse IgM)
e)Goat anti-mouse IgM-FITC(Santa Cruz sc-2082)
f)Goat anti-mouse IgG-FITC(Santa Cruz)
8, material
A) 4 porocyte culture plates (NUNC, #176740)
B) 75cm2 angle culturing bottle (NUNC, #156472)
9, plant and instrument
A) Biohazard Safety Equipment
B) CO2gas incubator
C) whizzer
D) flow cytometer
E) microscope
Embodiment 1 cell cultures
One, purpose: LD-1 is directly used in the hES cell cultures, observes its growing state and morphological change, and set up positive controls and negative control group simultaneously, compare synchronously.
Two, grouping and method: will be inoculated into the 26 generation hES cells (H1) that conditioned medium is cultivated on Matrigel in 4 orifice plates with Matrigel bag quilt, and be divided into three groups:
Cultivate (positive control) with conditioned medium for first group;
Use LD-1 culture medium culturing (that is: ordinary culture medium+40ng/ml Human FGF basic+500ng/ml Recombinant Mouse Noggin/Fc Chimera) for second group;
Cultivate (negative control) with ordinary culture medium for the 3rd group.
Press 0.5ml substratum/hole, change substratum every day once, passed once generation in 5-7 days, go down to posterity altogether 6 times.
Three, result: first group and second group of cell to the are during six generations, and growing state is still good, does not all have obvious differentiating phenomenon; The 3rd group of cell is at the first-generation existing obvious differentiating phenomenon in the time of the 5th day, and when reaching the s-generation, cell attachment reduces, and poor growth is broken up more obviously, can not go down to posterity again.
Four, conclusion: with LD-1 the hES cell of cultivating and hES cell no significant difference on morphology of cultivating with conditioned medium.
Embodiment 2 immune group chemistry detects
One, purpose:, detect the feature of some embryonic stem cells of cell after LD-1 cultivated for 6 generations with immune group chemistry method.
Two, method
1. cell: LD-1 cultivated and the hES cell seeding through repeatedly going down to posterity in 4 holes of 4 orifice plates, the hES cell that conditioned medium and ordinary culture medium are cultivated is planted respectively in 4 holes of two other 4 orifice plate simultaneously;
2. fixed cell: with above-mentioned cell cultures to the 4 days, absorb nutrient solution, add 4% secondary formaldehyde, placed 10 minutes under the room temperature, can be with cell fixation;
3. cell grouping: the aforementioned fixation cell is divided into 4 groups, and every group contains each 1 hole of hES cell that LD-1, conditioned medium and ordinary culture medium are cultivated respectively.
4. first antibody: a) Oct-4 (Chemicon MAB4305, mouse IgGl), b) SSEA-4 (Chemicon MAB4304, mouse IgG3), c) TRA-1-60 (Chemicon MAB4360, mouse IgM), d) TRA-1-81 (ChemiconMAB4381, mouse IgM)
5. second antibody: a) Goat anti-mouse IgM-FITC (Santa Cruz sc-2082), b) Goat anti-mouseIgG-FITC (Santa Cruz)
Three, result
| SSEA | TRA-1-60 | TRA-1-81 | |
| First group | +++ | +++ | +++ |
| Second group | +++ | +++ | +++ |
| The 3rd group | -/+ | -/+ | -/+ |
Annotate: +++strong positive; Negative or the weak positive of-/+.
Four, conclusion
The hES cell (first group) that hES cell (second group) that the LD-1 nutrient solution is cultivated and conditioned medium are cultivated is no significant difference in the immunofluorescence dyeing experiment of these above-mentioned three kinds of antibody.
Embodiment 3 quantitative analyses
One, purpose: use flow cytometer, the hES cell that LD-1 was cultivated after 6 generations carries out quantitative analysis.
Two, material is from the hES cell of first, second and third group, anti-Oct4 etc.
Three, method digests the back with 4 ℃ of resuspended 15min of PBS (containing 10% lowlenthal serum) with trypsin/EDTA.Add behind the anti-Oct4 monoclonal antibody 4 ℃ and hatch 30min, it is anti-to add FITC fluorescein-labeled two again.Upflowing cell instrument (Beckman Ku Erte Epics XL) detects, and data are by Ku Erte SYSTEM II software analysis.
Four, first and second group hES cell Oct4 positive rate all reaches more than 85% as a result; The 3rd group of hES cell Oct4 positive rate then is lower than 20%
Five, the hES cell of first and second group of conclusion all can be kept its undifferentiated state basically.
Embodiment 4 Analytical Chemical Experiment
One, purpose: the hES cell was divided into embryo internal layer, mesoderm and ectoderm histocyte respectively after LD-1 cultivated for 6 generations, thereby the hES cell of proof after LD-1 cultivates still has normal differentiation potential.
Two, material 1) anti-ectoderm antibody NFH (neurofilament heavy chain), Keratin15;
2) anti-mesoderm antibody T gene, beta-globin;
3) anti-entoderm antibody A FP (a-fetoprotein), amylase
Three, method is not broken up the hES cell colony with dispase digestion picking from the Matrigel glue, and renewed vaccination is in the culture dish of fibronectin bag quilt.Cultivate 12～14days with different nutrient solutions (with DMEM/F12 20% foetal calf serum, 1mM glutamine, 1% nonessential amino acid 0.1mM beta-mercaptoethanol are the basis) and impel its differentiation.
Four, the antibody mediated immunity fluorescent reaction of above-mentioned three germinal layers of result experiment all has positive reaction.
Five, the hES cell of conclusion LD-1 nutrient solution cultivation has kept the totipotency of its differentiation potential.
Embodiment 5 karyotypings
One, purpose: get rid of the possibility that LD-1 induces the hES cell carcinogenesis.
Two, (KaryoMax Colcemid solution GIBCO) handled 2～3 hours method hES cell with 0.1ug/ml Colcemid.Be resuspended in the 0.075M KCl solution to simplification with trysinization again, under 37 ℃, left standstill 20 minutes, use formaldehyde and acetate (3: 1) fixing again, carry out karyotyping with the G-banding method of standard then.
Three, the cell caryogram of cell that the result detects is 22xy.
Four, the hES cell that conclusion detected is the normal not human body cell of canceration.
The experiment of embodiment 6LD-1 medium optimization
One, purpose: the minimum effective concentration of bFGF and Noggin among the searching LD-1, to reduce cost.
Two, method respectively the concentration of bFGF among the LD-1 and Noggin is set at 4,8,10,20, behind 40ng/ml and 100,250,500, the 1000ng/ml, the growing state of observation of cell.
Three, it is the most satisfied that the result contains in the LD-1 substratum of Noggin of the bFGF of 40ng/ml and 500ng/ml the cell growing state.
Four, the concentration of bFGF and Noggin should be respectively 40ng/ml and 500ng/ml in the conclusion LD-1 substratum.
Claims (3)
1. people's embryonic stem cells substratum, feature are to add 10-40ng/ml bFGF and 250-500ng/ml Noggin in common hES cell culture medium.
2. the described people's embryonic stem cells of claim 1 substratum, feature are to add 40ng/ml bFGF and 500ng/ml Noggin in common hES cell culture medium.
3. prepare the purposes of the substratum of backer's embryonic stem cells growth with bFGF and Noggin, consumption is: 10-40ng/mlbFGF, 250-500ng/ml Noggin.
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Citations (6)
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| US5166065A (en) * | 1988-08-04 | 1992-11-24 | Amrad Corporation Limited | In vitro propagation of embryonic stem cells |
| JP2001508302A (en) * | 1997-01-10 | 2001-06-26 | ライフ テクノロジーズ，インコーポレイテッド | Embryonic stem cell serum replacement |
| CN1389565A (en) * | 2002-07-08 | 2003-01-08 | 徐如祥 | Culture process of human nerve stem cell |
| CN1416345A (en) * | 2000-03-09 | 2003-05-07 | 威斯康星校友研究基金会 | Serum free cultivation of primate embryonic stem cells |
| CN1416462A (en) * | 2000-01-11 | 2003-05-07 | 杰龙公司 | Techniques for growth and differentiatition of human pluripotent stem cells |
| CN1452655A (en) * | 2000-09-12 | 2003-10-29 | 加藤幸夫 | Method of culturing mesenchymal stem cells |
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5166065A (en) * | 1988-08-04 | 1992-11-24 | Amrad Corporation Limited | In vitro propagation of embryonic stem cells |
| JP2001508302A (en) * | 1997-01-10 | 2001-06-26 | ライフ テクノロジーズ，インコーポレイテッド | Embryonic stem cell serum replacement |
| CN1416462A (en) * | 2000-01-11 | 2003-05-07 | 杰龙公司 | Techniques for growth and differentiatition of human pluripotent stem cells |
| CN1416345A (en) * | 2000-03-09 | 2003-05-07 | 威斯康星校友研究基金会 | Serum free cultivation of primate embryonic stem cells |
| CN1452655A (en) * | 2000-09-12 | 2003-10-29 | 加藤幸夫 | Method of culturing mesenchymal stem cells |
| CN1389565A (en) * | 2002-07-08 | 2003-01-08 | 徐如祥 | Culture process of human nerve stem cell |
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