CN1570086A - Use and application of rat cardiac muscle cell cultured clear liquid - Google Patents
Use and application of rat cardiac muscle cell cultured clear liquid Download PDFInfo
- Publication number
- CN1570086A CN1570086A CNA031417256A CN03141725A CN1570086A CN 1570086 A CN1570086 A CN 1570086A CN A031417256 A CNA031417256 A CN A031417256A CN 03141725 A CN03141725 A CN 03141725A CN 1570086 A CN1570086 A CN 1570086A
- Authority
- CN
- China
- Prior art keywords
- cell
- substratum
- cardiac muscle
- embryonic stem
- rat
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the usage of rat cardiac muscle cell cultured supernatant. The rat cardiac muscle cell cultured supernatant can be used for culture medium for proliferation with non-differentiation of rat embryonic stem cell and its preparation method. The rat cardiac muscle cell cultured supernatant can be used exclusively or by combination of auxiliary materials for rat embryonic stem cell special culture substrate. The preparation method for the culture substrate is also provided. To culture rat embryonic stem cell for proliferation without differentiation in vitro culture by means of the method can derive a better effect than leukemia inhibiting factor(LIF). The invention employs rat cardiac cell, has low cost and simple preparation process, being superior to LIF in promoting ES cell wall sticking, proliferating and cloning.
Description
One, technical field
The present invention relates to the purposes of rat cardiac muscle cell cultured supernatant liquid and keep the mouse embryonic stem cell not break up and the substratum of breeding and preparation method thereof, especially rat cardiac muscle cell cultured supernatant liquid is used to prepare substratum and the preparation method who keeps the mouse embryonic stem cell not break up and breed.
Two, background technology
Embryonic stem cell (embryonic stem cell, ES cell) is the cell with all-round differentiation potential, and vitro culture is very easily broken up, and needs to add cytokine to suppress its differentiation.The method of vitro culture ES cell is concluded two classes: feeder layer culture method and no feeder layer culture method are arranged.It is early stage method that the feeder layer culture method is arranged, and mainly uses mouse embryo fibroblasts (MEF) or STO clone as feeder layer cells, treated termination its division after be prepared into the raising individual layer, with the ES cell seeding on such individual layer.MEF and STO can both secrete the factor-leukaemia inhibitory factor of promoting ES hyperplasia and suppressing its autonomous differentiation (leukemia inhibitory factor, LIF), content is at 500～1000IU/ml (secretor type); On MEF and STO matrix, also there is simultaneously LIF (matrix type).No feeder layer culture method is that the LIF that adds reorganization in basic medium is prepared into conditioned medium.Use feeder layer to cultivate the ES cell, have a lot of shortcomings: 1, MEF life is limited, can not go down to posterity for a long time external.Oversize in the subculture in vitro separately time, its ability that produces the short hyperplasia factor and inhibition differentiation factor can weaken even lose; 2, in the ES cell cultivation process, dead MEF or STO cell chromosome discharge sudden change that may cause the ES cell and the maintenance that influences normal karyotype.Therefore at present conditioned mediums that contain LIF that use no feeder layer make the ES cell grow in mode undifferentiated, adherent, cell colony more.
Leukaemia inhibitory factor (LIF), U.S. Pat 5166065 has concrete description to this, LIF is a U.S. CHEMICON house journal product, it is the cytokine of reorganization, not only cost an arm and a leg, the market price is 2600～3000 Renminbi/10 μ g, and according to Products Show concentration, can not keep the ES cell in external propagation, 2 times, 3 times of need or greater concn just can reach requirement.
Three, summary of the invention
The technical problem to be solved in the present invention provides a kind of cheap, effective, can keep the mouse embryonic stem cell not produce and break up and the substratum of propagation, utilize LIF to add the shortcoming of mouse ES cell culture medium vitro culture mouse ES cell cost height and poor effect to solve prior art in vitro culture.For this reason, the present invention takes following technical scheme:
A kind of purposes of rat cardiac muscle cell cultured supernatant, described rat cardiac muscle cell cultured supernatant can be used as the substratum that keeps the mouse embryonic stem cell not break up and breed.The substratum that rat cardiac muscle cell cultured supernatant not only can be bred as keeping the mouse embryonic stem cell not break up separately also can add the special culture media that suitable substratum auxiliary material becomes the mouse embryonic stem cell in rat cardiac muscle cell cultured supernatant.
A kind of substratum that keeps the mouse embryonic stem cell not break up and breed, described substratum contains rat cardiac muscle cell cultured supernatant and substratum auxiliary material.Here the mouse myocardial cell culture supernatant of being said is exactly to get myocardial cell's culture supernatant that rat heart is prepared by certain method.
In order to make the mouse embryonic stem cell reach better effect in external growth, rat cardiac muscle cell cultured supernatant liquid and glutamine preparation are being combined into the substratum that can keep the mouse embryonic stem cell not break up and breed, and the final concentration of glutamine in substratum is 1～4mmol/L.
Same rat cardiac muscle cell cultured supernatant liquid and non-essential amino acid also can be mixed with the substratum that composition is bred as keeping the mouse embryonic stem cell not break up, and the final concentration of non-essential amino acid is 0.1mmol/L.
But rat cardiac muscle cell cultured supernatant liquid and final concentration are 1～4mmol/L glutamine and final concentration is also common combination of 0.1mmol/L non-essential amino acid, becomes the substratum that can keep the mouse embryonic stem cell not break up and breed.
The substratum of the mouse embryonic stem cell that above-mentioned these three kinds of modes are combined into, the 2 mercapto ethanol that also can add content respectively and be 0.05mmol/L constitutes the more perfect mouse ES cell culture medium of assembly separately.
In order to make substratum maintain the sufficient nutrition material, also can in substratum, add foetal calf serum, the content of foetal calf serum is 10%～20%, is that final concentration is that the glutamine of 1～4mmol/L and content are that 10%～20% foetal calf serum is formed and can be kept the mouse embryonic stem cell not break up the substratum of breeding as can add content in rat cardiac muscle cell cultured supernatant liquid.
In the prior art, be in nutrient solution, to add LIF, can make mouse embryonic stem cell (mouse ES cell) keep a kind of undifferentiated self state, we find in experiment, rat cardiac muscle cell cultured supernatant liquid can keep not breaking up when being used alone as mouse ES cell culture medium and breed, and its cell growth state is than only far better with LIF, and adherent, the propagation and the clone that are more conducive to the ES cell form; Also can keep mouse ES cell not break up when cultivating and breed uniting, and effect is better than independent use LIF with LIF.When rat cardiac muscle cell cultured supernatant liquid and LIF united as substratum, the content of LIF selected for use 1000 units/ml (being unit/ml) more suitable.For guaranteeing to keep the mouse embryonic stem cell not break up the effect of the substratum of breeding, usually select for use 2～3 age in week rat myocardial cell 1～6 foster supernatant liquor of being commissioned to train, the common quality of rat cardiac muscle cell cultured supernatant liquid that this situation obtains is better, can guarantee that mouse ES cell does not produce differentiation external can propagation rapidly.
A kind of method that is used to prepare the substratum that keeps the mouse embryonic stem cell not break up and breed, the preparation method of described rat cardiac muscle cell cultured supernatant carries out in the following order:
A. get cleaning level SD rat, put to death, it is dirty to core, and removes pericardium and clot, reticular tissue, and washing shreds;
B. small tissue blocks is moved in the culturing bottle and cultivate, substratum is: contain the DMEM substratum of 10%～20% bovine serum, including final concentration is 1～4mmol/L glutamine, PH7.2～7.4; This is first-generation myocardial cell, is designated as G1, when cell grow to bottle floorage 70%～80% the time, go down to posterity, be designated as G2, so go down to posterity, collect each for myocardial cell's culture supernatant, in-30 ℃, standby.
For turning out better rat cardiac muscle cell cultured supernatant liquid.Can behind the step a of aforesaid method, increase digestion step: m. the cardiac scissors of handling well is minced with 0.02%-0.25%EDTA-trypsinase at 37 ℃ of digestion 15min, during constantly vibration gently, washing, centrifugal, abandon supernatant, keep sedimentation cell and small tissue blocks.Carry out the b step again.
Bovine serum among the preparation method of described substratum is available foetal calf serum.
The washing of mentioning in the preparation process of mouse myocardial cell culture supernatant with the damping fluid washing of no calcium magnesium, is washed as the phosphoric acid salt buffer usually.
The present invention compared with prior art has following advantage and effect: (1) rat cardiac muscle cell cultured supernatant liquid can directly be used as mouse ES cell culture medium, need not add other component.(2) cultivate mouse ES cell with the present invention, make mouse ES cell keep not breaking up and breeding in vitro culture, effect is better than LIF.The description of product that provides according to LIF, during mouse ES cell cultures, the LIF optimal concentration is 1000 units/ml (being 1000unit/ml), finds in the experiment that this concentration can not make mouse ES cell keep the undifferentiated proliferation mode, needing increases LIF concentration, and the present invention makes the ES cell in-vitro growth in order.(3) the present invention's rat myocardial cell that has drawn from, cost is low, and the LIF price is comparatively expensive.(4) the present invention not only prepares simply, and is better than LIF at aspects such as promoting ES cell attachment, increment, clone's formation.
Four, description of drawings
Fig. 1 is the mouse embryonic stem cell of culture medium culturing that embodiment 1 joins: mouse ES cell colony (4 * 10 times)
Fig. 2 is the mouse embryonic stem cell of culture medium culturing that embodiment 2 joins: mouse ES cell colony (10 * 10 times)
Fig. 3 is the mouse embryonic stem cell of culture medium culturing that embodiment 2 joins: mouse ES cell colony (4 * 10 times)
Five, embodiment
Embodiment 1
1.1 prepare following material:
1.1.1 mouse embryonic stem cell line (mouse embryonic stem cell) is so kind as to give by Robertson doctor E..
1.1.2 rat cardiac muscle cell cultured supernatant is provided for oneself
1.1.3 glutamine (L-glutamine, the worker is given birth in Shanghai, the packing of Amresco product, LotNo.1490B40).
1.1.4 foetal calf serum
1.1.5 the Dulbecco`s Modified Eagle Medium of Gibco company (DMEM) high glucose medium
1.1.6 EDTA-trypsinase
1.1.7 0.1% gelatin solution: gelatin (gelatin, Huamei Bio-Engrg Co.,, Lot No.0205) is made into 0.1%, 0.22 μ m membrane filtration after the heat a little with tri-distilled water, and 4 ℃ of preservations are standby.
1.1.8 alkaline phosphatase staining agents useful for same: naphthols AS-MX phosphoric acid ester; Dimethyl formamide is given birth to the worker available from Shanghai; 2-amino-2-methyl-1,3 propylene glycol, the import packing of Shanghai chemical reagents corporation; Fast blue RR is available from Fluka company.
1.1.9 laboratory animal: the SD rat is provided by Zhejiang Province's Experimental Animal Center.
1.2 the preparation method is as follows:
1.2.1 rat cardiac muscle cell cultured supernatant preparation
The disconnected neck of rat in 3 ages in week is put to death,, take out heart, remove pericardium and clot, reticular tissue, wash, remove blood, shred with phosphate buffered saline buffer (be PBS, pH transfers to 7.4) with 75% alcohol-pickled 10 minutes.Digest 15min with 0.02%-0.25%EDTA-trypsinase at 37 ℃, constantly vibration gently during this time, adding 10 times of amount PBS washs, 1200 left the heart 5 minutes, abandon supernatant, adopt paster method in 37 ℃ with cultivating myocardial cell's nutrient solution in sedimentation cell and the small tissue blocks immigration culturing bottle, 5%CO
2The middle cultivation.Substratum is: (including final concentration is 2mmol/L glutamine (glutamin), PH7.2～7.4 for fetal bovine serum, DMEM high glucose medium FBS) to contain 15% foetal calf serum.This is first-generation myocardial cell, is designated as G1, when cell grow to bottle floorage 70%～80% the time, go down to posterity, be designated as G2, collect the nutrient solution of 1-6 for cell, the centrifugal 8min of 1200rpm, supernatant filters with 0.22 μ m, packing ,-20 ℃ of preservations are standby.
1.2.2 the culture plate pre-treatment adds 0.1% gelatin solution in 24 well culture plates, be advisable at the bottom of covering full hole, places 30min in the room temperature, inhales and abandons gelatin solution, and solution residual in the culture plate is dried up, whole process is noted aseptic technique.With the culture plate sealing, standby, can place for 2 weeks for 4 ℃.The culture plate of handling like this can make cell more adherent when cultivating.
The culture medium preparation of breeding 1.2.3 can keep the mouse embryonic stem cell not break up:
Culture medium preparation, getting rat cardiac muscle cell cultured supernatant adding final concentration is the 2mmol/L glutamine, final concentration is 15% foetal calf serum mixing, constitutes substratum, i.e. mouse ES substratum.
1.3 mouse ES cell cultures recovery ES cell is with the pretreated culture plate culturing cell of gelatin, 37 ℃, 5%CO
2The middle cultivation after 24 hours changed liquid, and amount was changed liquid in later per 24 hours half.Should note the ES cell is blown and beaten into individual cells during inoculation ES, but because the existence irritation cell of ES cell mass differentiation.
1.4 checking: adopt the azo coupling reaction method to carry out alkaline phosphatase staining (AKP dyeing).Do not break up the ES cell AKP positive (cell is dyed blue look); Noble cells AKP feminine gender (cell is dyed yellow).
Above-mentioned cultured mouse ES cell is placed on microscopically and observes.
1.5 result
It is all very fast that microscopically is observed above-mentioned ES cell growth, and 24～48h can be observed little ES cell colony formation, and cell volume is little, and nuclear is big, and the caryoplasm ratio is big, and cell is arranged closely.The AKP stained positive.Still can present the colony growth after passing for 6 generations, AKP measures still positive.
Mouse ES cell colony upgrowth situation as shown in Figure 1.
Embodiment 2
2.1 prepare following material:
2.1 prepare following material:
2.1.1 mouse embryonic stem cell line (mouse embryonic stem cell) is so kind as to give by Robertson doctor E..
2.1.2 rat cardiac muscle cell cultured supernatant is provided for oneself
2.1.3 glutamine (L-glutamine, the worker is given birth in Shanghai, the packing of Amresco product, LotNo.1490B40).
2.1.4 the Dulbecco`s Modified Eagle Medium of Gibco company (DMEM) high glucose medium
2.1.5 foetal calf serum
2.1.6 EDTA-trypsinase
2.1.7 0.1% gelatin solution: gelatin (gelatin, Huamei Bio-Engrg Co.,, Lot No.0205) is made into 0.1%, 0.22 μ m membrane filtration after the heat a little with tri-distilled water, and 4 ℃ of preservations are standby.
2.1.8 alkaline phosphatase staining agents useful for same: naphthols AS-MX phosphoric acid ester; Dimethyl formamide is given birth to the worker available from Shanghai; 2-amino-2-methyl-1,3 propylene glycol, the import packing of Shanghai chemical reagents corporation; Fast blue RR is available from Fluka company.
2.1.9 laboratory animal: the SD rat is provided by Zhejiang Province's Experimental Animal Center.
2.1.10 15%FCS-DMEM, contain leukaemia inhibitory factor 1000unit/ml (recombinantmurine leukemia inhibitory factor, rmLIF, available from Chemicon, LotNo.11161032; )
2.2 the preparation method is as follows:
2.2.1 rat cardiac muscle cell cultured supernatant preparation
The disconnected neck of the rat in 2 ages in week is put to death,, take out heart, remove pericardium and clot, reticular tissue, with phosphate buffered saline buffer (be PBS, pH transfers to 7.4) washing, remove blood, shred.Digest 15min with 0.02%-0.25%EDTA-trypsinase at 37 ℃, constantly vibration gently during this time, adding 10 times of amount PBS washs, 1200 left the heart 5 minutes, abandon supernatant, adopt paster method in 37 ℃ with cultivating myocardial cell's nutrient solution in sedimentation cell and the small tissue blocks immigration culturing bottle, 5%CO
2The middle cultivation.Substratum is: (fetal bovine serum, DMEM high glucose medium FBS), final concentration are 2mmol/L glutamine (glutamin), PH7.2～7.4 to contain 15% foetal calf serum.This is first-generation myocardial cell, is designated as G1, when cell grow to bottle floorage 70%～80% the time, go down to posterity, be designated as G2, collect the nutrient solution of 1-6 for cell, the centrifugal 8min of 1200rpm, supernatant filters with 0.22 μ m, packing ,-30 ℃ of preservations are standby.
2.2.2 the culture plate pre-treatment adds 0.1% gelatin solution in 24 well culture plates, be advisable at the bottom of covering full hole, places 30min in the room temperature, inhales and abandons gelatin solution, and solution residual in the culture plate is dried up, whole process is noted aseptic technique.With the culture plate sealing, standby, can place for 2 weeks for 4 ℃.The culture plate of handling like this can make cell more adherent when cultivating.
The culture medium preparation of breeding 2.2.3 can keep the mouse embryonic stem cell not break up:
Culture medium preparation, getting rat cardiac muscle cell cultured supernatant adding final concentration is the 3mmol/L glutamine, and content is 10% foetal calf serum, and content is that the LIF of 1000unit/ml mixes, and constitutes substratum, i.e. mouse ES substratum.
2.3 mouse ES cell cultures recovery ES cell is with the pretreated culture plate culturing cell of gelatin, 37 ℃, 5%CO
2The middle cultivation after 24 hours changed liquid, changes liquid later on every other day.Should note the ES cell is blown and beaten into individual cells during inoculation ES, but because the existence irritation cell of ES cell mass differentiation.
2.4 checking: adopt the azo coupling reaction method to carry out alkaline phosphatase staining (AKP dyeing).Do not break up the ES cell AKP positive (cell is dyed blue look); Noble cells AKP feminine gender (cell is dyed yellow).
Above-mentioned cultured mouse embryonic stem cell is placed on microscopically and observes.
2.5 result
It is all very fast that microscopically is observed above-mentioned ES cell growth, and 24～48h can be observed little ES cell colony formation, and cell volume is little, and nuclear is big, and the caryoplasm ratio is big, and cell is arranged closely.The AKP stained positive.Still can present the colony growth after passing for 6 generations, AKP measures still positive.
Mouse ES cell colony upgrowth situation such as Fig. 2, shown in Figure 3.
Embodiment 3
3.1 prepare following material:
3.1.1 mouse embryonic stem cell line (mouse embryonic stem cell) is so kind as to give by Robertson doctor E..
3.1.2 rat cardiac muscle cell cultured supernatant is provided for oneself
3.1.3 non-essential amino acid (non-essential amino acids solution, Gibco company, Lot No.1127865)
3.1.4 the Dulbecco`s Modified Eagle Medium of Gibco company (DMEM) high glucose medium
3.1.5 foetal calf serum
3.1.6 0.1% gelatin solution: gelatin (gelatin, Huamei Bio-Engrg Co.,, Lot No.0205) is made into 0.1%, 0.22 μ m membrane filtration after the heat a little with tri-distilled water, and 4 ℃ of preservations are standby.
3.1.7 alkaline phosphatase staining agents useful for same: naphthols AS-MX phosphoric acid ester; Dimethyl formamide is given birth to the worker available from Shanghai; 2-amino-2-methyl-1,3 propylene glycol, the import packing of Shanghai chemical reagents corporation; Fast blue RR is available from Fluka company.
3.1.8 laboratory animal: the SD rat is provided by Zhejiang Province's Experimental Animal Center.
3.2 the preparation method is as follows:
3.2.1 rat cardiac muscle cell cultured supernatant preparation
The disconnected neck of the rat in 3 ages in week is put to death, take out heart, remove pericardium and clot, reticular tissue,, remove blood, shred with phosphate buffered saline buffer (be PBS, pH transfers to 7.4) washing.Small tissue blocks moved into cultivate myocardial cell's nutrient solution in the culturing bottle and adopt paster method, 5%CO in 37 ℃
2The middle cultivation.Substratum is: (including final concentration is 3mmol/L glutamine (glutamin), PH7.2～7.4 for fetal bovine serum, DMEM high glucose medium FBS) to contain 15% foetal calf serum.This is first-generation myocardial cell, is designated as G1, when cell grow to bottle floorage 70%～80% the time, go down to posterity, be designated as G2, collect the nutrient solution of 1-6 for cell, the centrifugal 8min of 1200rpm, supernatant filters with 0.22 μ m, packing ,-30 ℃ of preservations are standby.
3.2.2 the culture plate pre-treatment adds 0.1% gelatin solution in 24 well culture plates, be advisable at the bottom of covering full hole, places 30min in the room temperature, inhales and abandons gelatin solution, and solution residual in the culture plate is dried up, whole process is noted aseptic technique.With the culture plate sealing, standby, can place for 2 weeks for 4 ℃.The culture plate of handling like this can make cell more adherent when cultivating.
The culture medium preparation of breeding 3.2.3 can keep the mouse embryonic stem cell not break up:
Culture medium preparation, getting rat cardiac muscle cell cultured supernatant adding final concentration is the 0.1mmol/L non-essential amino acid, final concentration is 20% foetal calf serum mixing, constitutes substratum, i.e. mouse ES substratum.
3.3 mouse ES cell cultures recovery ES cell is with the pretreated culture plate culturing cell of gelatin, 37 ℃, 5%CO
2The middle cultivation changed liquid in 24 hours, changed liquid later on every other day.Should note the ES cell is blown and beaten into individual cells during inoculation ES, but because the existence irritation cell of ES cell mass differentiation.
3.4 checking: adopt the azo coupling reaction method to carry out alkaline phosphatase staining (AKP dyeing).Do not break up the ES cell AKP positive (cell is dyed blue look); Noble cells AKP feminine gender (cell is dyed yellow).
Above-mentioned cultured mouse embryonic stem cell is placed on microscopically and observes.
3.5 result
It is all very fast that microscopically is observed above-mentioned ES cell growth, and 24～48h can be observed little ES cell colony formation, and cell volume is little, and nuclear is big, and the caryoplasm ratio is big, and cell is arranged closely.The AKP stained positive.Still can present the colony growth after passing for 6 generations, AKP measures still positive.
Embodiment 4
4.1 prepare following material:
4.1.1 mouse embryonic stem cell line (mouse embryonic stem cell) is so kind as to give by Robertson doctor E..
4.1.2 rat cardiac muscle cell cultured supernatant is provided for oneself
4.1.3 glutamine (L-glutamine, the worker is given birth in Shanghai, the packing of Amresco product, LotNo.1490B40).
4.1.4 the Dulbecco`s Modified Eagle Medium of Gibco company (DMEM) substratum
4.1.5 foetal calf serum
4.1.6 2 mercapto ethanol
4.1.7 alkaline phosphatase staining agents useful for same: naphthols AS-MX phosphoric acid ester; Dimethyl formamide is given birth to the worker available from Shanghai; 2-amino-2-methyl-1,3 propylene glycol, the import packing of Shanghai chemical reagents corporation; Fast blue RR is available from Fluka company.
4.1.8 laboratory animal: the SD rat is provided by Zhejiang Province's Experimental Animal Center.
4.2 the preparation method is as follows:
4.2.1 rat cardiac muscle cell cultured supernatant preparation
The disconnected neck of the rat in 3 ages in week is put to death, take out heart, remove pericardium and clot, reticular tissue,, remove blood, shred with phosphate buffered saline buffer (be PBS, pH transfers to 7.4) washing.Small tissue blocks moved into cultivate myocardial cell's nutrient solution in the culturing bottle and adopt paster method, 5%CO in 37 ℃
2The middle cultivation.Substratum is: (including final concentration is 4mmol/L glutamine (glutamin), PH7.2～7.4 for fetal bovine serum, DMEM substratum FBS) to contain 15% foetal calf serum.This is first-generation myocardial cell, is designated as G1, when cell grow to bottle floorage 70%～80% the time, go down to posterity, be designated as G2, collect the nutrient solution of 1-6 for cell, the centrifugal 8min of 1200rpm, supernatant filters with 0.22 μ m, packing ,-30 ℃ of preservations are standby.
The culture medium preparation of breeding 4.2.2 can keep the mouse embryonic stem cell not break up:
Culture medium preparation, getting rat cardiac muscle cell cultured supernatant adding final concentration is the 0.1mmol/L non-essential amino acid, and final concentration is 20% foetal calf serum, and final concentration is that the 0.05mmol/L 2 mercapto ethanol mixes, and constitutes substratum, i.e. mouse ES substratum.
4.3 mouse ES cell cultures recovery ES cell is with the pretreated culture plate culturing cell of gelatin, 37 ℃, 5%CO
2The middle cultivation after 24 hours changed liquid, changes liquid later on every other day.Should note the ES cell is blown and beaten into individual cells during inoculation ES, but because the existence irritation cell of ES cell mass differentiation.
4.4 checking: adopt the azo coupling reaction method to carry out alkaline phosphatase staining (AKP dyeing).Do not break up the ES cell AKP positive (cell is dyed blue look); Noble cells AKP feminine gender (cell is dyed yellow).
Above-mentioned cultured mouse embryonic stem cell is placed on microscopically and observes.
4.5 result
Microscopically is observed all very fast 48h of above-mentioned ES cell growth and be can be observed little ES cell colony formation, and cell volume is little, and nuclear is big, and the caryoplasm ratio is big, and cell is arranged closely.The AKP stained positive.Still can present the colony growth after passing for 6 generations, AKP measures still positive.
Embodiment 5
5.1 prepare following material:
5.1.1 mouse embryonic stem cell line (mouse embryonic stem cell) is so kind as to give by Robertson doctor E..
5.1.2 rat cardiac muscle cell cultured supernatant is provided for oneself
5.1.3 glutamine (L-glutamine, the worker is given birth in Shanghai, the packing of Amresco product, LotNo.1490B40).
5.1.4 the Dulbecco`s Modified Eagle Medium of Gibco company (DMEM) high glucose medium
5.1.5 EDTA-trypsinase
5.1.6 0.1% gelatin solution: gelatin (gelatin, Huamei Bio-Engrg Co.,, Lot No.0205) is made into 0.1%, 0.22 μ m membrane filtration after the heat a little with tri-distilled water, and 4 ℃ of preservations are standby.
5.1.7 alkaline phosphatase staining agents useful for same: naphthols AS-MX phosphoric acid ester; Dimethyl formamide is given birth to the worker available from Shanghai; 2-amino-2-methyl-1,3 propylene glycol, the import packing of Shanghai chemical reagents corporation; Fast blue RR is available from Fluka company.
5.1.8 laboratory animal: the SD rat is provided by Zhejiang Province's Experimental Animal Center.
5.2 the preparation method is as follows:
5.2.1 rat cardiac muscle cell cultured supernatant preparation
The disconnected neck of rat in 2 ages in week is put to death,, take out heart, remove pericardium and clot, reticular tissue, wash, remove blood, shred with phosphate buffered saline buffer (be PBS, pH transfers to 7.4) with 75% alcohol-pickled 10 minutes.Digest 15min with 0.02%-0.25%EDTA-trypsinase at 37 ℃, constantly vibration gently during this time, adding 10 times of amount PBS washs, 1200 left the heart 5 minutes, abandon supernatant, adopt paster method in 37 ℃ with cultivating myocardial cell's nutrient solution in sedimentation cell and the small tissue blocks immigration culturing bottle, 5%CO
2The middle cultivation.Substratum is: (including final concentration is 2mmol/L glutamine (glutamin), PH7.2～7.4 for fetal bovine serum, DMEM high glucose medium FBS) to contain 15% foetal calf serum.This is first-generation myocardial cell, is designated as G1, when cell grow to bottle floorage 70%～80% the time, go down to posterity, be designated as G2, collect the nutrient solution of 1-6 for cell, the centrifugal 8min of 1200rpm, supernatant filters with 0.22 μ m, packing ,-20 ℃ of preservations are standby.
5.2.2 the culture plate pre-treatment adds 0.1% gelatin solution in 24 well culture plates, be advisable at the bottom of covering full hole, places 30min in the room temperature, inhales and abandons gelatin solution, and solution residual in the culture plate is dried up, whole process is noted aseptic technique.With the culture plate sealing, standby, can place for 2 weeks for 4 ℃.The culture plate of handling like this can make cell more adherent when cultivating.
The culture medium preparation of breeding 5.2.3 can keep the mouse embryonic stem cell not break up:
Culture medium preparation, the rat cardiac muscle cell cultured supernatant that gets collection is directly as mouse ES substratum.
5.3 mouse ES cell cultures recovery ES cell is with the pretreated culture plate culturing cell of gelatin, 37 ℃, 5%CO
2The middle cultivation after 24 hours changed liquid, changes liquid later on every other day.Should note the ES cell is blown and beaten into individual cells during inoculation ES, but because the existence irritation cell of ES cell mass differentiation.
5.4 checking: adopt the azo coupling reaction method to carry out alkaline phosphatase staining (AKP dyeing).Do not break up the ES cell AKP positive (cell is dyed blue look); Noble cells AKP feminine gender (cell is dyed yellow).
Above-mentioned cultured mouse embryonic stem cell is placed on microscopically and observes.
5.5 result
It is all very fast that microscopically is observed above-mentioned ES cell growth, and 24～48h can be observed little ES cell colony formation, and cell volume is little, and nuclear is big, and the caryoplasm ratio is big, and cell is arranged closely.The AKP stained positive.
Claims (12)
1. the purposes of a rat cardiac muscle cell cultured supernatant is characterized in that the substratum that described rat cardiac muscle cell cultured supernatant is bred as keeping the mouse embryonic stem cell not break up.
2. a substratum that keeps the mouse embryonic stem cell not break up and breed is characterized in that described substratum contains rat cardiac muscle cell cultured supernatant and substratum auxiliary material.
3. the substratum that keeps the mouse embryonic stem cell not break up and breed as claimed in claim 2 is characterized in that described substratum is the composition of one of rat cardiac muscle cell cultured supernatant and following formula or following formula.1. final concentration be 1～4mmol/L glutamine 2. final concentration be the 0.1mmol/L non-essential amino acid
4. the substratum that keeps the mouse embryonic stem cell not break up and breed as claimed in claim 3 is characterized in that it is the 0.05mmol/L 2 mercapto ethanol that described substratum also contains final concentration.
5. as claim 3 or the 4 described substratum that keep the mouse embryonic stem cells not break up and breed, it is characterized in that described substratum also contains 10%～20% foetal calf serum.
6. the substratum that keeps the mouse embryonic stem cell not break up and breed as claimed in claim 5 is characterized in that described substratum also contains the LIF that final concentration is 1000 units/ml.
7. as the described substratum that can keep the mouse embryonic stem cell not break up and breed of one of claim 1-4, it is characterized in that described rat adopted for 2～3 ages in week.
8. maintenance as claimed in claim 7 mouse embryonic stem cell does not break up and the substratum of breeding, it is characterized in that described rat cardiac muscle cell cultured supernatant be 1～6 generation rat cardiac muscle cell cultured supernatant.
9. preparation method who is used to prepare substratum as claimed in claim 2 is characterized in that the preparation method of described rat cardiac muscle cell cultured supernatant carries out in the following order:
A. get cleaning level SD rat, put to death, it is dirty to core, and removes pericardium and clot, reticular tissue, and washing shreds;
B. small tissue blocks is moved in the culturing bottle and cultivate, substratum is: contain the DMEM substratum of 10%～20% bovine serum, include final concentration 1～4mmol/L glutamine, PH7.2～7.4; This is first-generation myocardial cell, is designated as G1, when cell grow to bottle floorage 70%～80% the time, go down to posterity, be designated as G2, so go down to posterity, collect each for myocardial cell's culture supernatant, in-20 ℃～-30 ℃ refrigerations, standby.
The preparation method of 10 substratum as claimed in claim 9 is characterized in that the preparation method of described rat cardiac muscle cell cultured supernatant increases digestion step behind step a:
M. the cardiac scissors of handling well minces with 0.02%～0.25%EDTA-trypsinase at 37 ℃ of digestion 15min, during constantly vibrate gently, wash, centrifugal, abandon supernatant, reservation sedimentation cell and small tissue blocks.
11. the preparation method of substratum as claimed in claim 9 is characterized in that described bovine serum is a foetal calf serum.
12., it is characterized in that the damping fluid washing of described washing with no calcium magnesium as the preparation method of one of claim 9-11 described substratum.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA031417256A CN1570086A (en) | 2003-07-18 | 2003-07-18 | Use and application of rat cardiac muscle cell cultured clear liquid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA031417256A CN1570086A (en) | 2003-07-18 | 2003-07-18 | Use and application of rat cardiac muscle cell cultured clear liquid |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1570086A true CN1570086A (en) | 2005-01-26 |
Family
ID=34471035
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA031417256A Pending CN1570086A (en) | 2003-07-18 | 2003-07-18 | Use and application of rat cardiac muscle cell cultured clear liquid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1570086A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9458429B2 (en) | 2010-06-01 | 2016-10-04 | Pias Corporation | Mesenchymal stem cell attractant and method for attracting mesenchymal stem cell |
-
2003
- 2003-07-18 CN CNA031417256A patent/CN1570086A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9458429B2 (en) | 2010-06-01 | 2016-10-04 | Pias Corporation | Mesenchymal stem cell attractant and method for attracting mesenchymal stem cell |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Yang et al. | Tissue engineering of cementum/periodontal-ligament complex using a novel three-dimensional pellet cultivation system for human periodontal ligament stem cells | |
| CN1452655A (en) | Method of culturing mesenchymal stem cells | |
| Kuznetsov et al. | In vivo bone formation by progeny of human embryonic stem cells | |
| US7935527B2 (en) | Methods for culturing human embryonic stem cells | |
| CN1186977C (en) | Crustacean larva raising method and apparatus | |
| CN1104844C (en) | Oral compsn. for animals | |
| CN1571835A (en) | Keratinocytes which may be used as biologically active substances for the treatment of wounds | |
| CN1636054A (en) | Mesenchymal cells and osteoblasts from human embryonic stem cell | |
| CN1735685A (en) | Treatment of tissue with undifferentiated mesenchymal cells | |
| CN1649999A (en) | Avian cell lines for the production of useful substances | |
| CN102127522A (en) | Human umbilical mesenchymal stem cell and preparation method thereof | |
| CN101056974A (en) | Identifying and separating pluripotent cell from non bone cartilage mesenchymal tissue | |
| CN1391605A (en) | Gynogenetic or androgenetic production of pluripotent cells and cell lines, and use thereof to produce differentiated cells and tissues | |
| CN1860222A (en) | Stem cells for clinical and commercial uses | |
| CN1872025A (en) | Production for skin beautification and care, and preparation method | |
| CN1708582A (en) | Composition for culturing multipotent stem cells and utilization of the same | |
| CN1075636A (en) | Promote the method for growth of animal and the powder composition of the microbial cell of the antibacterial that contains the Clostridium that kills | |
| CN1778905A (en) | Separating culture and use for fatty mesenchymal dry cell | |
| CN101037669A (en) | Method for inducing liver cell from human embryonic stem cells | |
| CN1800371A (en) | In vitro maturing culture method for human immature ovum | |
| CN1269956C (en) | Non serum substratum for in vitro culture and amplification of cutaneous keratin cell | |
| CN1649998A (en) | Method for growth of human conjunctival tissue equivalents for research, clinical ocular surface transplantation and tissue engineering | |
| CN1570086A (en) | Use and application of rat cardiac muscle cell cultured clear liquid | |
| CN1958781A (en) | Reagent peptone, and preparation method | |
| CN103122334A (en) | Primary culture and purification method of intestinal epithelial cells of carassius auratus gibelio |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |