CN101654667A - Method for establishing lactation model of cow mammary gland epithelial cells - Google Patents
Method for establishing lactation model of cow mammary gland epithelial cells Download PDFInfo
- Publication number
- CN101654667A CN101654667A CN200910072961A CN200910072961A CN101654667A CN 101654667 A CN101654667 A CN 101654667A CN 200910072961 A CN200910072961 A CN 200910072961A CN 200910072961 A CN200910072961 A CN 200910072961A CN 101654667 A CN101654667 A CN 101654667A
- Authority
- CN
- China
- Prior art keywords
- cell
- mammary gland
- frozen
- hplc
- epithelial cells
- 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
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a method for establishing a lactation model of cow mammary gland epithelial cells for researching the milk protein gene expression regulation mechanism and providing favorable conditions for testing a mammary gland expression vector. The testing method comprises the following steps: carrying out primary culture and identification of the cow mammary gland epithelial cells; determining the secretion situation of lactose by using the HPLC method; determining the secretion situation of beta-casein protein by using the HPLC method; and carrying out cryopreservation and recovery on the cells. The method adopts the tissue block method to culturing the cow mammary gland epithelial cells in different periods, compared with other primary culture methods of the mammary gland epithelial cells, such as enzyme digestion and the like, the method does not affect the activity of the mammary gland epithelial cells, the culture cost is very low, and the operation is very simple. The method realizes the continuation of normal culture of a cow mammary gland epithelial cell system, thereby property and effectively preserving a large number of precious cow mammary gland tissue materials. The method provides the important testing materials and a technical platform for researching the lactation mechanism of the cow mammary gland during the different development periods.
Description
(1) technical field
The present invention relates to biocytology, is exactly a kind of method of setting up lactation model of cow mammary gland epithelial cells specifically.
(2) background technology
Mammary epithelial cell is the basic structure and the functional unit of lactation, by studying lactation Expression of Related Genes in it, can how the mammary specific expression gene be activated in order to disclose, how initial sum is kept lactation to lactation critical function gene, regulation and control milk component provides theoretical foundation.Simultaneously, help to set up a kind of mammary gland biological modulated control techniques of regulating and control milk bovine lactation critical function gene expression dose and significantly improving milk yield.
Former be commissioned to train to support be meant that taking out histocyte in the body begins to cultivate period before going down to posterity for the first time, be a distinctive inevitable growth phase.Cell or tissue has been finished transition and the adaptive process from internal milieu to external environment, has recovered division growth and the ability of growing.The former foster mammary epithelial cell of being commissioned to train has synthetic and lactescent specific function, it is the target cell of making galactophore biological reactor, the mammary epithelial cell with synthetic and secretion milk-protein of vitro culture can antimer in the secretion capacity (Xu Manni etc., 2003) of mammary gland.The tissue that derives from mammary gland lactation period has kept the biological function of self because of it, and has half point class epithelial stem cell group's feature.Therefore to lactation period the mammary epithelial cell vitro culture research be the basis that makes up cell lactation model system.
There is the mammary epithelial cell system of lactation function both can be used for studying milk protein gene expression and lactation mechanism (Wheeler etc., 1995), can be used as the valuable materials that research dried breast phase apoptosis and mammary gland are degenerated again.Simultaneously can also be as the detection system of mammary gland specific expression vector, estimate the validity and the reasonableness (Xu Manni etc. of mammary gland specific expression vector, 2003), the cell model that is further used as galactophore biological reactor is produced valuable biological activity protein (Li Zhen, 1997).It is the research tool that has using value that the mammary epithelial cell system of lactation function is arranged.C127, T47D derive from the secreting function (Hu Yanqiu etc., 2006) that clone such as mouse, people and other most clones have been lost milk-protein, are not suitable as research lactation mechanism and detect the cell model of mammary gland expression vector.At present, the clone that rarely seen KIM22, HC11, CID9 etc. derive from mouse, bovine mammary gland has milk-protein synthesis capability (Katrina etc., 2000), and these clones also just are used for laboratory study now, do not form commercial prod.
Mammary epithelial cell is well differentiated somatocyte, and there is certain difficulty in its vitro culture, and suitable culture system is crucial to the foundation of mammary epithelial cell system.Vitro culture technology for cow mammary gland epithelial cells is immature always.
(3) summary of the invention
The object of the present invention is to provide a kind of technology platform of the bovine lactation Mechanism Study of suckling, the method for setting up lactation model of cow mammary gland epithelial cells of favourable condition is provided for the detection of the research of milk protein gene expression and regulation mechanism and mammary gland expression vector.
The object of the present invention is achieved like this: the described method of setting up lactation model of cow mammary gland epithelial cells, and the experimental technique step is as follows:
Step 1: former being commissioned to train of cow mammary gland epithelial cells supported and evaluation
The sterilization of breast middle part cuts a small amount of mammary tissue piece according to routine operation, also peels off fatty tissue and reticular tissue with D-Hanks liquid cleansing tissue piece immediately as far as possible, gets the mammary gland acinar tissue and partly is cut into 1mm
3Fritter, be inoculated in the Tissue Culture Flask that overlays collagen protein with the spacing of 0.5cm, be paved with whole bottle after, be inverted and be incubated at 37 ℃ CO
23～4h in the incubator, when tissue block firmly is affixed on the collagen, gently grown cultures liquid is added in the culturing bottle, be as the criterion to cover tissue block, continue in the incubator to cultivate with being placed on, change a nutrient solution every 2d, reach at 80%～90% o'clock to the cell stand density, utilize mammary epithelial cell and inoblast that the different pair cells of trypsinase susceptibility are gone down to posterity and cultivate to obtain the cow mammary gland epithelial cells of purifying; Adopt conventional cellular immunofluorescence dyeing process that specific Keratin sulfate 18 antibody of mammary epithelial cell are carried out fluorescent dye, carry out laser co-focusing and observe so that mammary epithelial cell is identified;
Step 2: the HPLC method is measured the secretion situation of lactose
The processing of test sample: with the lactose standard model of moving phase dissolving different concns, with the resuspended different treatment group of pure water cell, place and use homogenizer Mechanical Crushing cell on ice, the HPLC that is used for lactose behind the 0.22 μ m membrane filtration lysate detects; The testing conditions of HPLC: chromatographic column, octadecylsilane chemically bonded silica, Φ 4.6 * 150mm; Column temperature, room temperature; The ultraviolet detection wavelength, 195nm; Moving phase, 5% second eyeball is through 0.22 μ m membrane filtration, ultrasonic degas; Flow velocity, constant current 0.6ml/min; Sampling volume, 10 μ l; The lactose standard substance that with concentration are 1mg/ml, 5mg/ml, 10mg/ml, 15mg/ml, 20mg/ml respectively carry out HPLC, are X-coordinate with the lactose concn, and peak area is that ordinate zou is made the lactose typical curve; Cell sample and lactose standard substance adopt identical HPLC testing conditions;
Step 3: the HPLC method is measured the secretion situation of beta-casein
The processing of test sample: with the beta-casein standard model of moving phase dissolving different concns, with the resuspended different treatment group of pure water cell, place and use homogenizer Mechanical Crushing cell on ice, the HPLC that is used for beta-casein behind the 0.22 μ m membrane filtration lysate detects; The testing conditions of HPLC: tsk gel post, Φ 7.8 * 300mm; Column temperature, room temperature; The ultraviolet detection wavelength, 280nm; Moving phase, ultrapure water is through 0.22 μ m membrane filtration, ultrasonic degas; Flow velocity, constant current 0.6ml/min; Sampling volume, 10 μ l; The beta-casein standard substance that with concentration are 1mg/ml, 5mg/ml, 10mg/ml, 15mg/ml, 20mg/ml respectively carry out HPLC, are X-coordinate with the lactose concn, and peak area is that ordinate zou is made the beta-casein typical curve; Cell sample and beta-casein standard substance adopt identical HPLC testing conditions;
Step 4: the frozen and recovery of cell
As frozen buffer protection liquid, carry out frozen with 1: 1 ratio to mammary epithelial cell with 20%DF12 nutrient solution, 15%DMSO, HEPES and Sodium.alpha.-ketopropionate with FBS; Concrete operation method is: discard nutrient solution, digest mammary epithelial cell with 0.25% trypsinase/EDTA, add nutrient solution and stop digestion, make cell suspension, collect in the centrifuge tube, the centrifugal cell precipitation that gets of 1000r/min; With the FBS re-suspended cell of 37 ℃ of preheatings, be sub-packed in the frozen pipe with an amount of density, in each frozen pipe, dropwise add the frozen damping fluid of 4 ℃ of precoolings then, gently mixing; To be affixed on the label of cow mammary gland epithelial cells title, frozen date and nutrient solution type on the frozen pipe; In mid--80 ℃ of refrigerators of incubation chamber, make cell slowly frozen frozen duct occlusion, behind the 12-24h cell is dropped into the liquid nitrogen container midium or long term and preserve; Following method is adopted in the recovery of cell: take out the frozen pipe of preserving cell, put into melting fully of 37 ℃～40 ℃ of water-baths, in case ice cube disappears at once its taking-up; Clean frozen pipe outside to reduce opportunities for contamination with 75% ethanol; The frozen pipe content of sucking-off in centrifuge tube, slowly add fresh medium and gently mixing to clean cell; 1000r/min, centrifugal 5min, cell precipitation, according to the cell density before frozen with cell precipitation with the resuspended inoculation of fresh medium.
The present invention can provide technology platform for the research of milk cow lactation Study on Mechanism and milk yield raising, and the research that is established as the milk protein gene expression and regulation mechanism of mammary epithelial cell system and the detection of mammary gland expression vector provide favourable condition.The present invention adopts tissue block method that the cow mammary gland epithelial cells of different times is cultivated and has all obtained success.Other mammary epithelial cells cultured method of former generation such as this method and enzymic digestion are compared, and it does not influence the activity of mammary epithelial cell, and it is very cheap to cultivate cost, operates very easy.The culture system of the cow mammary gland epithelial cells that the present invention sets up be DF12 grown cultures liquid wherein add 10% homemade foetal calf serum and 5 μ g/ml Regular Insulin in 37 ℃, 5%CO
2Condition is cultivated, and it requires lower to culture condition, and cost is low, very is suitable for the growth of mammary epithelial cell.It is frozen that the present invention adopts cells frozen storing liquid with strong buffering and protective capability and slow frozen method that cow mammary gland epithelial cells is carried out repeatedly, and the cell after the recovery has higher survival rate.Realized the continuity of the cow mammary gland epithelial cells system of normal cultivation by the present invention, thereby made the cow mammary gland organization material of a large amount of preciousnesses obtain properly effectively to preserve.The present invention provides important experiment material and technology platform for the research of cow mammary gland different development stage lactation mechanism.
(4) description of drawings
Fig. 1 is the inverted microscope observations of different development stage mammary epithelial cell vitro culture of the present invention;
Fig. 2 is that the Keratin sulfate 18 of the cow mammary gland epithelial cells of purifying of the present invention is identified;
Fig. 3 is the lactose HPLC of the present invention lactose standard substance of A:50mg/ml as a result;
Fig. 4 is lactose HPLC of the present invention B as a result: the lactose secretion situation of cell;
Fig. 5 is the HPLC beta-casein standard substance of A:50mg/ml as a result of beta-casein of the present invention;
Fig. 6 is the HPLC B as a result of beta-casein of the present invention: the beta-casein secretion situation of cell.
(5) embodiment
The invention will be further described for example below in conjunction with accompanying drawing.
Embodiment 1: a kind of method of setting up lactation model of cow mammary gland epithelial cells of the present invention, and the experimental technique step is as follows:
Step 1: former being commissioned to train of cow mammary gland epithelial cells supported and evaluation
The sterilization of breast middle part cuts a small amount of mammary tissue piece according to routine operation, also peels off fatty tissue and reticular tissue with D-Hanks liquid cleansing tissue piece immediately as far as possible, gets the mammary gland acinar tissue and partly is cut into 1mm
3Fritter, be inoculated in the Tissue Culture Flask that overlays collagen protein with the spacing of 0.5cm, be paved with whole bottle after, be inverted and be incubated at 37 ℃ CO
2About 3～4h in the incubator, when tissue block firmly is affixed on the collagen, gently grown cultures liquid is added in the cultivation, be as the criterion to cover tissue block, continue in the incubator to cultivate with being placed on, change a nutrient solution every 2d, reach at 80%～90% o'clock to the cell stand density, utilize mammary epithelial cell and inoblast that the different pair cells of trypsinase susceptibility are gone down to posterity and cultivate to obtain the cow mammary gland epithelial cells of purifying; Adopt conventional cellular immunofluorescence dyeing process that specific Keratin sulfate 18 antibody of mammary epithelial cell are carried out fluorescent dye and carry out the laser co-focusing observation so that mammary epithelial cell is identified;
Step 2: the HPLC method is measured the secretion situation of lactose
The processing of test sample: with the lactose standard model of moving phase dissolving different concns, with the resuspended different treatment group of pure water cell, place and use homogenizer Mechanical Crushing cell on ice, the HPLC that is used for lactose behind the 0.22 μ m membrane filtration lysate detects; The testing conditions of HPLC: chromatographic column, octadecylsilane chemically bonded silica, Φ 4.6 * 150mm; Column temperature, room temperature; The ultraviolet detection wavelength, 195nm; Moving phase, 5% second eyeball is through 0.22 μ m membrane filtration, ultrasonic degas; Flow velocity, constant current 0.6ml/min; Sampling volume, 10 μ l; The lactose standard substance that with concentration are 1mg/ml, 5mg/ml, 10mg/ml, 15mg/ml, 20mg/ml respectively carry out HPLC, are X-coordinate with the lactose concn, and peak area is that ordinate zou is made the lactose typical curve; Cell sample and lactose standard substance adopt identical HPLC testing conditions;
Step 3: the HPLC method is measured the secretion situation of beta-casein
The processing of test sample: with the beta-casein standard model of moving phase dissolving different concns, with the resuspended different treatment group of pure water cell, place and use homogenizer Mechanical Crushing cell on ice, the HPLC that is used for beta-casein behind the 0.22 μ m membrane filtration lysate detects; The testing conditions of HPLC: tsk gel post, Φ 7.8 * 300mm; Column temperature, room temperature; The ultraviolet detection wavelength, 280nm; Moving phase, ultrapure water is through 0.22 μ m membrane filtration, ultrasonic degas; Flow velocity, constant current 0.6ml/min; Sampling volume, 10 μ l; The beta-casein standard substance that with concentration are 1mg/ml, 5mg/ml, 10mg/ml, 15mg/ml, 20mg/ml respectively carry out HPLC, are X-coordinate with the lactose concn, and peak area is that ordinate zou is made the beta-casein typical curve; Cell sample and beta-casein standard substance adopt identical HPLC testing conditions;
Step 4: the frozen and recovery of cell
As frozen buffer protection liquid, carry out frozen with 1: 1 ratio to mammary epithelial cell with 20%DF12 nutrient solution, 15%DMSO, HEPES and Sodium.alpha.-ketopropionate with FBS; Concrete operation method is: discard nutrient solution, digest mammary epithelial cell with 0.25% trypsinase/EDTA, add nutrient solution and stop digestion, make cell suspension, collect in the centrifuge tube, the centrifugal cell precipitation that gets of 1000r/min; With the FBS re-suspended cell of 37 ℃ of preheatings, be sub-packed in the frozen pipe with an amount of density, in each frozen pipe, dropwise add the frozen damping fluid of 4 ℃ of precoolings then, gently mixing; To be affixed on the label of cow mammary gland epithelial cells title, frozen date and nutrient solution type on the frozen pipe; In mid--80 ℃ of refrigerators of incubation chamber, make cell slowly frozen frozen duct occlusion, behind the 12-24h cell is dropped into the liquid nitrogen container midium or long term and preserve; Following method is adopted in the recovery of cell: take out the frozen pipe of preserving cell, put into melting fully of 37 ℃～40 ℃ of water-baths, in case ice cube disappears at once its taking-up; Clean frozen pipe outside to reduce opportunities for contamination with 75% ethanol; The frozen pipe content of sucking-off in centrifuge tube, slowly add fresh medium and gently mixing to clean cell; 1000r/min, centrifugal 5min, cell precipitation, according to the cell density before frozen with cell precipitation with the resuspended inoculation of fresh medium.
Embodiment 2: in conjunction with Fig. 1-Fig. 6, and experiment that the present invention does:
Experiment material: healthy holstein cow mammary tissue.The mammary tissue of sampling is in 2 months pubescence, 7 days lactation periods, 280 days lactation periods respectively, does 3 days phases of breast, 10 days perinatal periods.Experiment reagent: mouse tail collagen protein, the friend is given birth in Hangzhou; High-quality foetal calf serum, DF12 substratum, Gibco company; Cytokeratin 18 antibody, German Acris company; The PI dyestuff, Regular Insulin, Sigma; Hplc grade methanol, Tianjin recovery; The lactose standard substance, Military Medical Science Institute; The beta-casein standard substance, Sigma.Experimental installation: ZHJH-1112 vertical current Bechtop, Shanghai ZHICHENG Anaiytical Instrument Manufacturing Co., Ltd.; CO-150 type CO
2Incubator, Japan; The automatic dual pure water distiller of SZ-93, Shanghai Yarong Biochemical Instrument Plant; The LC-10AT high performance liquid chromatograph, day island proper Tianjin, SPD-10A UV-detector; The DFC280 inverted phase contrast microscope, German LEICA; TC SP2AOBS laser scanning co-focusing microscope, German LEICA.
Experimental result is judged; The expression that is positive of the immunofluorescence dyeing of the cow mammary gland epithelial cells Keratin sulfate 18 of separation and purification, isolation identification cow mammary gland epithelial cells.By improved cell cryopreservation of the present invention and method for resuscitation, successful acquisition has also been preserved a large amount of lactational cow mammary gland epithelial cells, and HPLC detects and shows that this cell has the secretion capacity of lactose and beta-casein.
The different times cow mammary gland epithelial cells is cultivated the observation of situation:
The present invention chooses the vitro culture of lactational holstein cow tissue block method mammary epithelial cell, every 3d changes nutrient solution 1 time, begin to occur inoblast when being cultured to about 9d around the mammary tissue piece, mammary epithelial cell appears when being cultured to 10～12d left and right sides, epithelioid cell and fibroblast-like cells be mixed growth mutually, but majority is an epithelial cell, and the black part among the figure is divided into mammary tissue piece (as Fig. 1).To enzymic digestion susceptibility is different the two is separated with inoblast according to epithelial cell.In the former generation or passage cell culture of mixed growth, add 0.25% tryptic D-Hanks liquid earlier, 37 ℃ of digestion are observed under inverted microscope, treat that inoblast breaks away from when cultivating diapire, stop digestion, and inclining the inoblast that major part comes off.Remaining mammary tissue piece and mammary epithelial cell on every side thereof place incubator to continue to cultivate.2～3 times the epithelium posterius cell is able to purifying.To 90% at the bottom of epithelial cell is paved with bottle when above, with ordinary method to the mammary epithelial cell cultivation of going down to posterity.By inverted phase contrast microscope the cow mammary gland epithelial cells of different times is carried out morphological observation.The mammary gland cell of purifying is the epithelioid cell, is polygon, and the form homogeneous is arranged between the cell closely, and the milk cow in lactation period mammary epithelial cell of the vitro culture that this research obtains is rich in the emulsion droplet (Fig. 1) that differs in size.
The evaluation of cow mammary gland epithelial cells Keratin sulfate 18:
The present invention uses the dyeing immunofluorescence cell method and has detected the skelemin of mammary epithelial cell behind the purifying---the expression of cytokeratin 18.The expression of the mammary epithelial cell Keratin sulfate 18 that laser co-focusing observations display separation is cultivated be positive (Fig. 2).Partly for being expressed in the Keratin sulfate 18 in the tenuigenin, red is the painted nucleus of PI to the green fluorescence that shows among the figure, proves that it is the cow mammary gland epithelial cells of purifying.
The lactose excretory HPLC of mammary epithelial cell detects
Fig. 3, Fig. 4 are lactose standard substance and mammary epithelial cell lactose excretory detected result.Fig. 3 is the HPLC collection of illustrative plates of lactose standard substance.Main peak retention time in the examination cell sample color atlas is consistent with lactose standard substance main peak retention time, illustrates that mammary epithelial cell has the ability (Fig. 4) of secretion lactose.
The lactose excretory HPLC of mammary epithelial cell detects
Fig. 5 is beta-casein standard substance and mammary epithelial cell beta-casein excretory detected result.Fig. 5 is the HPLC collection of illustrative plates of beta-casein standard substance.Main peak retention time in the examination cell sample color atlas is consistent with beta-casein standard substance main peak retention time, illustrates that mammary epithelial cell has excreting beta-caseic ability (Fig. 6).
Claims (1)
1. method of setting up lactation model of cow mammary gland epithelial cells, it is characterized in that: the experimental technique step is as follows:
Step 1: former being commissioned to train of cow mammary gland epithelial cells supported and evaluation
The sterilization of breast middle part cuts a small amount of mammary tissue piece according to routine operation, also peels off fatty tissue and reticular tissue with D-Hanks liquid cleansing tissue piece immediately as far as possible, gets the mammary gland acinar tissue and partly is cut into 1mm
3Fritter, be inoculated in the Tissue Culture Flask that overlays collagen protein with the spacing of 0.5cm, be paved with whole bottle after, be inverted and be incubated at 37 ℃ CO
23～4h in the incubator, when tissue block firmly is affixed on the collagen, gently grown cultures liquid is added in the culturing bottle, be as the criterion to cover tissue block, continue in the incubator to cultivate with being placed on, change a nutrient solution every 2d, reach at 80%～90% o'clock to the cell stand density, utilize mammary epithelial cell and inoblast that the different pair cells of trypsinase susceptibility are gone down to posterity and cultivate to obtain the cow mammary gland epithelial cells of purifying; Adopt conventional cellular immunofluorescence dyeing process that specific Keratin sulfate 18 antibody of mammary epithelial cell are carried out fluorescent dye and carry out the laser co-focusing observation so that mammary epithelial cell is identified;
Step 2: the HPLC method is measured the secretion situation of lactose
The processing of test sample: with the lactose standard model of moving phase dissolving different concns, with the resuspended different treatment group of pure water cell, place and use homogenizer Mechanical Crushing cell on ice, the HPLC that is used for lactose behind the 0.22 μ m membrane filtration lysate detects; The testing conditions of HPLC: chromatographic column, octadecylsilane chemically bonded silica, Φ 4.6 * 150mm; Column temperature, room temperature; The ultraviolet detection wavelength, 195nm; Moving phase, 5% second eyeball is through 0.22 μ m membrane filtration, ultrasonic degas; Flow velocity, constant current 0.6ml/min; Sampling volume, 10 μ l; The lactose standard substance that with concentration are 1mg/ml, 5mg/ml, 10mg/ml, 15mg/ml, 20mg/ml respectively carry out HPLC, are X-coordinate with the lactose concn, and peak area is that ordinate zou is made the lactose typical curve; Cell sample and lactose standard substance adopt identical HPLC testing conditions;
Step 3: the HPLC method is measured the secretion situation of beta-casein
The processing of test sample: with the beta-casein standard model of moving phase dissolving different concns, with the resuspended different treatment group of pure water cell, place and use homogenizer Mechanical Crushing cell on ice, the HPLC that is used for beta-casein behind the 0.22 μ m membrane filtration lysate detects; The testing conditions of HPLC: tsk gel post, Φ 7.8 * 300mm; Column temperature, room temperature; The ultraviolet detection wavelength, 280nm; Moving phase, ultrapure water is through 0.22 μ m membrane filtration, ultrasonic degas; Flow velocity, constant current 0.6ml/min; Sampling volume, 10 μ l; The beta-casein standard substance that with concentration are 1mg/ml, 5mg/ml, 10mg/ml, 15mg/ml, 20mg/ml respectively carry out HPLC, are X-coordinate with the lactose concn, and peak area is that ordinate zou is made the beta-casein typical curve; Cell sample and beta-casein standard substance adopt identical HPLC testing conditions;
Step 4: the frozen and recovery of cell
As frozen buffer protection liquid, carry out frozen with 1: 1 ratio to mammary epithelial cell with 20%DF12 nutrient solution, 15%DMSO, HEPES and Sodium.alpha.-ketopropionate with FBS; Concrete operation method is: discard nutrient solution, digest mammary epithelial cell with 0.25% trypsinase/EDTA, add nutrient solution and stop digestion, make cell suspension, collect in the centrifuge tube, the centrifugal cell precipitation that gets of 1000r/min; With the FBS re-suspended cell of 37 ℃ of preheatings, be sub-packed in the frozen pipe with an amount of density, in each frozen pipe, dropwise add the frozen damping fluid of 4 ℃ of precoolings then, gently mixing; To be affixed on the label of cow mammary gland epithelial cells title, frozen date and nutrient solution type on the frozen pipe; In mid--80 ℃ of refrigerators of incubation chamber, make cell slowly frozen frozen duct occlusion, behind the 12-24h cell is dropped into the liquid nitrogen container midium or long term and preserve; Following method is adopted in the recovery of cell: take out the frozen pipe of preserving cell, put into melting fully of 37 ℃～40 ℃ of water-baths, in case ice cube disappears at once its taking-up; Clean frozen pipe outside to reduce opportunities for contamination with 75% ethanol; The frozen pipe content of sucking-off in centrifuge tube, slowly add fresh medium and gently mixing to clean cell; 1000r/min, centrifugal 5min, cell precipitation, according to the cell density before frozen with cell precipitation with the resuspended inoculation of fresh medium.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910072961A CN101654667A (en) | 2009-09-23 | 2009-09-23 | Method for establishing lactation model of cow mammary gland epithelial cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910072961A CN101654667A (en) | 2009-09-23 | 2009-09-23 | Method for establishing lactation model of cow mammary gland epithelial cells |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101654667A true CN101654667A (en) | 2010-02-24 |
Family
ID=41709122
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200910072961A Pending CN101654667A (en) | 2009-09-23 | 2009-09-23 | Method for establishing lactation model of cow mammary gland epithelial cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101654667A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101857852A (en) * | 2010-05-31 | 2010-10-13 | 东北农业大学 | Method for building dairy cattle breast acinus lactation model in vitro |
| CN103146639A (en) * | 2013-02-26 | 2013-06-12 | 内蒙古农业大学 | Culture medium for accelerating in vitro differentiation of mammary epithelial cells of dairy cattle and application thereof |
| CN105316279A (en) * | 2014-07-17 | 2016-02-10 | 北京大北农科技集团股份有限公司动物医学研究中心 | Method for efficiently separating and purifying mammary epithelial cells |
| CN106035315A (en) * | 2016-04-21 | 2016-10-26 | 中国农业科学院兰州畜牧与兽药研究所 | Cryopreservation solution of mammary tissue of dairy cow for primary culture of mammary epithelial cells of dairy cow, and application of cryopreservation solution in cryopreservation method for mammary tissue of dairy cow |
| CN111214879A (en) * | 2019-11-01 | 2020-06-02 | 上海蓓蕊医疗科技有限公司 | Cell negative pressure filtering device and use method thereof |
-
2009
- 2009-09-23 CN CN200910072961A patent/CN101654667A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101857852A (en) * | 2010-05-31 | 2010-10-13 | 东北农业大学 | Method for building dairy cattle breast acinus lactation model in vitro |
| CN103146639A (en) * | 2013-02-26 | 2013-06-12 | 内蒙古农业大学 | Culture medium for accelerating in vitro differentiation of mammary epithelial cells of dairy cattle and application thereof |
| CN103146639B (en) * | 2013-02-26 | 2015-08-19 | 内蒙古农业大学 | Promote substratum and the application thereof of cow mammary gland epithelial cells vitro differentiation |
| CN105316279A (en) * | 2014-07-17 | 2016-02-10 | 北京大北农科技集团股份有限公司动物医学研究中心 | Method for efficiently separating and purifying mammary epithelial cells |
| CN106035315A (en) * | 2016-04-21 | 2016-10-26 | 中国农业科学院兰州畜牧与兽药研究所 | Cryopreservation solution of mammary tissue of dairy cow for primary culture of mammary epithelial cells of dairy cow, and application of cryopreservation solution in cryopreservation method for mammary tissue of dairy cow |
| CN106035315B (en) * | 2016-04-21 | 2018-07-10 | 中国农业科学院兰州畜牧与兽药研究所 | Cow mammary gland tissue freezing solution for cow mammary gland epithelial cells original cuiture and its application in breast tissue cryopreservation methods |
| CN111214879A (en) * | 2019-11-01 | 2020-06-02 | 上海蓓蕊医疗科技有限公司 | Cell negative pressure filtering device and use method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Spiliotis et al. | Axenic in vitro cultivation of Echinococcus multilocularis metacestode vesicles and the generationof primary cell cultures | |
| CN101942413A (en) | Birth defect cell bank and construction method thereof | |
| CN101818127B (en) | Method for separating and culturing mouse primitive spermatogonia | |
| CN102344906B (en) | Hair follicle stem cell separation culture method | |
| CN103525752B (en) | Separation and purification method of goat mammary epithelial cells | |
| CN105543164A (en) | Primary isolated culture method for dairy cow mammary epithelial cells | |
| CN101654667A (en) | Method for establishing lactation model of cow mammary gland epithelial cells | |
| CN109280636A (en) | A method of suspend domestication 293T cell | |
| CN102304492A (en) | Primary culture method for liver cells of crucian carp | |
| CN104178451B (en) | From milk, separate and cultivate method and the special culture media of cow mammary gland epithelial cells | |
| CN102978154A (en) | Primary culture method for simultaneously separating skin fibroblasts and epidermis cells of Inner mongolia white cashmere goat | |
| CN113293128A (en) | Hexagrammos otakii spermatogonia culture medium and culture method | |
| CN104877955A (en) | Method for separating and purifying rat dermal microvascular endothelium cells | |
| CN111454990A (en) | Human jugular auxiliary nerve ganglionic tumor immortalized cell strain and application thereof | |
| CN104894056A (en) | Method for constructing spleen tissue cell line of acipenser dabryanus | |
| CN104195100A (en) | In-vitro culture method of mammary gland epithelial cells | |
| Harkey et al. | Mass isolation and culture of sea urchin micromeres | |
| CN115786269A (en) | Method for constructing placenta organoid model | |
| CN104419682A (en) | Construction of trisomy 21 syndrome cell model and cell bank thereof by using recombined SV40LT and hTERT genes | |
| CN104419684A (en) | Construction of 18-trisomy syndrome cell model and cell bank of 18-trisomy syndrome cell by SV40LT gene transfection | |
| CN109055305A (en) | A kind of separating and extracting process of milk cow liver stem cells | |
| CN104419727A (en) | Construction of neural tube defect cell model and cell bank thereof under induction of hTERT recombinant | |
| CN117736975A (en) | Method for separating, extracting and culturing endometrial stem cells | |
| CN104419677A (en) | Construction of trisomy 18 syndrome cell model and cell bank thereof by hTERT transgenosis | |
| CN116751739A (en) | Primary separation method of human nourishing stem cells and special kit thereof |
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 |
Open date: 20100224 |