CN101407784B - Separation and purification method, as well as preservation method for ruminant galactophore epithelial cell - Google Patents
Separation and purification method, as well as preservation method for ruminant galactophore epithelial cell Download PDFInfo
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Abstract
The invention discloses a method for separating, purifying and culturing a mammary epithelial cell of a ruminant animal, which includes the following steps of: 1) the sampling of a mammary tissue; 2) the preparing of a culture fluid; 3) the culturing of a tissue block for obtaining a primary cultured mammary epithelial cell; 4) the purifying of the mammary epithelial cell for obtaining a purifiedmammary epithelial cell; and 5) the subculturing of the mammary epithelial cell. The invention simultaneously provides a preservation method for the mammary epithelial cell of the ruminant animal, which includes the following steps of: 1) preparing a freezing protection fluid which comprises the mammary epithelial cell of the ruminant animal; and 2) gradually and slowly reducing the temperature of the freezing protection fluid. The method can be adopted for prolonging the preservation time of the mammary epithelial cell of the ruminant animal.
Description
Technical field
The present invention relates to research fields such as tissue and cell engineering, transgenic animal, particularly to separation, purifying, cultivation and the frozen method of mammary epithelial cell.
Background technology
Mammary gland is important skin external secretion organ, has multiple physiology, biochemistry and immunologic function, can sufficient nutrient is provided and satisfy the demand of its growth for baby or cub.Giving full play to of these functions depends on a series of specific variations that mammary gland itself experiences: mammogenesis, mammary gland differentiation, lactation and mammary gland reconstruction and restoration of old ways etc.In fact, because the interaction of airframe systems and local numerous variable factors, make people have very big difficulty for the research of the growth of mammary epithelial cell, functional segregation, restoration of old ways, immune defense etc.The animal cell culture model is simulated in vivo environment and be widely used in fields such as biology, medical science, trophology, toxicology to a certain extent, can be used for studying cell form, grow, cytotrophy and microprocesses such as metabolism and pathology, carry out the research of aspects such as exogenous material mechanism of absorption, transporting pathway and the mechanism of action.Cultivate and the co-culture of cells pattern to simple single epithelial cell from the organ vitro culture of complexity, can well solve problem for specific factor research.Successively there is the scholar that the vitro culture of ruminant galactophore epithelial cell is studied both at home and abroad, but present research mainly rests on cultural method, morphology and the hormone aspects such as regulation and control to mammary gland cell, and for separation method, report is not quite similar both at home and abroad, separating effect differs, morphologic data is thorough not to the utmost, relates to that its immunity is identified, microtexture is observed, Study of Cryopreservation of Human is few, to the systematic study of its lactation function still less.
Summary of the invention
The technical problem to be solved in the present invention provides the separation and purification method of the effective ruminant galactophore epithelial cell of a kind of separation and purification, and a kind of store method that can prolong the ruminant galactophore epithelial cell of shelf time.
In order to solve the problems of the technologies described above, the invention provides a kind of separation, purifying culture method of ruminant galactophore epithelial cell, may further comprise the steps:
1), the sampling of mammary tissue:
Select the mammary gland that is separated with ruminating animal for use, the mammary gland of removing behind reticular tissue and the fat is divided into little block organization, and after sterilization and clean; Getting volume is 0.9～1.1mm
3The mammary tissue piece, standby;
2), preparation nutrient solution:
Nutrient solution obtains according to following feed ratio:
In the DMEM/F12 of every ml nutrient solution, add 1 μ g hydrocortisone, 5 μ g Transferrins,iron complexess, 5 μ g Regular Insulin, 5 μ g lactotropins, 10ng Urogastron, 2 μ mol glutamine, 100IU mycillin and 0.1ml foetal calf serum;
3), tissue block is cultivated:
The mammary tissue piece of step 1) gained is put into the cell culture container of spreading mouse tail collagen, put 37 ℃, 5%CO
2, cultivate under the saturated humidity condition, when cell covered 80% container bottom surface-area, the Digestive system that with mass concentration is 0.15% trypsinase and 0.02%EDTA was in 37 ℃ of above-mentioned attached cell 5-8min of digestion suspension; After the retraction of 80-90% cell under inverted microscope, change circle and intercellular substance enlarge, stop digesting with the D-Hanks liquid that contains 10% (volume percent) FBS immediately; The cell suspension of gained gets cell after filtration with centrifugal;
Clean above-mentioned cell 3 times with nutrient solution; Adjust cell density (adopting nutrient solution to dilute) with blood cell counting plate at last, with 5 * 10
4The density of/mL is implanted culture plate and is cultivated, and places 37 ℃, 5%CO
2, cultivate under the saturated humidity condition; When being cultured to cell and covering 80% plate bottom surface area, the former foster mammary epithelial cell of being commissioned to train;
4), the purifying of mammary epithelial cell:
With former be commissioned to train mammary epithelial cell foster and adopt difference enzyme process and adherent method repeatedly, in order to the purifying epithelial cell; Specific as follows:
Difference enzyme process: be that the Digestive system of 0.25% pancreatin is digested to fibrocyte 10min earlier, remove inoblast and Digestive system with mass concentration; Adding mass concentration again is the mixture slaking liquid digestive epithelium cell 3-5min of 0.15% pancreatin and 0.02%EDTA;
Adherent method repeatedly: after the epithelial cell that obtains adjusted cell density (adopting nutrient solution to dilute) with centrifugal, counting after filtration, with 5 * 10
4The density of/mL is planted in Tissue Culture Flask again, places 37 ℃, 5%CO
2, cultivate under the saturated humidity condition;
Treat that cell spreads at the bottom of the plate at 80% o'clock, repeat successively more above-mentioned poor enzyme process and repeatedly adherent method 1～2 this, obtain the mammary epithelial cell after prepurification is handled;
Mammary epithelial cell after above-mentioned prepurification is handled reaches 80% when converging, and removes nutrient solution; With no Ca
2+, Mg
2+Hanks liquid flushing cell, adding mass concentration then is 0.15% pancreatin and 0.02%EDTA mixture slaking liquid, with peptic cell,, use the D-Hanks liquid of 10%FBS to stop digestion until the retraction of 80-90% cell under inverted microscope, when becoming circle and intercellular substance expansion; Gained be mammary epithelial cell behind the purifying;
5), the cultivation of going down to posterity of mammary epithelial cell:
Mammary epithelial cell behind the above-mentioned purifying through centrifugal, is got cell, then clean cell 3 times with nutrient solution; Remove supernatant liquor, with nutrient solution suspension cell again, and with hemocyte plate counting adjustment cell density (adopting nutrient solution to dilute), with 5 * 10
4The density of/mL is implanted culturing bottle and is cultivated, and places 37 ℃, 5%CO
2, cultivate under the saturated humidity condition, the next day change liquid.
Improvement as separation of the present invention, purifying culture method: in the step 3), the distance of cultivating between the mammary tissue piece in the plate hole is 0.45～0.55cm; Cell suspension is the stainless steel filtering net filtration of 105 μ m with the aperture.
The present invention also provides a kind of store method of ruminant galactophore epithelial cell simultaneously, may further comprise the steps:
1), preparation frozen solution:
Frozen solution is made up of the component of following volume percent: 10%DMSO, 40% foetal calf serum and 50% cell suspension; Described cell suspension is the mixture that is formed by cell and DMEM/F12 basic medium, and the density of cell in cell suspension is 1 * 10
6/ mL, described cell are the former mammary epithelial cell of being commissioned to train foster mammary epithelial cell, the mammary epithelial cell behind the purifying or going down to posterity and cultivating;
2), with above-mentioned steps 1) the progressively cooling at a slow speed of the frozen solution of gained:
A, 4 ℃ reduce to-8 ℃, and rate of temperature fall is 1 ℃/min;-8 ℃ kept one minute;
B ,-8 ℃ reduce to-20 ℃, and rate of temperature fall is 0.5 ℃/min;
C ,-20 ℃ reduce to-120 ℃, and rate of temperature fall is 0.3 ℃/min;
D ,-120 ℃ frozen solution of above-mentioned steps gained is directly utilized liquid nitrogen cooling, be cooled to-196 ℃ until frozen solution;
Adopt liquid nitrogen freezing to preserve above-mentioned-196 ℃ frozen solution liquid.
The present invention is intended to set up the mammary epithelial cell lactation model that can be used for correlative studys such as ruminant milk mechanism of secretion, mammary gland physiology, breast phase apoptosis, mammary gland degeneration and mammary gland and nutrition supply, diseases prevention and treatment; Processes such as mammary epithelial cell separation, purifying, cultivation, growth, preservation have been contained in invention.
The separation and purification method of ruminant galactophore epithelial cell of the present invention, adopt tissue mass cell culture, process difference enzyme process (0.25% trysinization inoblast, 0.15% pancreatin and 0.02%EDTA digestive epithelium cell) and repeatedly the adherent method purifying epithelial cell, discovery is in the DMEM/F-12 nutrient solution that has added compositions such as Regular Insulin, lactotropin, Transferrins,iron complexes, cell is typical epithelium sample form, the cell that reached for the tenth generation molecular marker for increased proliferation that remains unchanged, growth rapidly.
In order to prove advantage of the present invention, the contriver has made following confirmatory experiment:
Experiment 1, contriver after to the purifying of step 4) gained mammary epithelial cell and the mammary epithelial cell of cultivating that goes down to posterity of step 5) gained carried out following evaluation, specific as follows:
According to the experimental procedure of routine immunization cytochemical methods, one anti-is anti-mouse cytokeratin 18 antibody of rabbit, and two anti-ly are goat anti-rabbit igg, through DAB colour developing, the epithelium characteristic of observing mammary gland cell.
The result finds as shown in Figure 3: the CK-18 specificity is expressed in mammary epithelial cell, and has detected identical result in the epithelial cell after going down to posterity, and illustrate that the mammary gland cell Keratin sulfate characteristic (epithelium characteristic) of cultivation still exists.
Experiment 2, contriver after to the purifying of step 4) gained mammary epithelial cell and the mammary epithelial cell of cultivating that goes down to posterity of step 5) gained carried out Ultrastructural observation, specific as follows:
Collection is gone down to posterity and is cultured to the 9th generation eugonic mammary gland cell (1-5 * 10
6Individual), the centrifugal 10min of 1000r/min, remove supernatant liquor, fixedly spend the night for 4 ℃ with cold 2.5% glutaraldehyde, through 0.1M phosphoric acid buffer rinsing sample three times, fix with 1% osmic acid again,, under transmission electron microscope, observe the ultrastructure of mammary gland cell after steps such as the dehydration of ethanol gradient, embedding, polymerization, section, dyeing are made ultrathin section(ing).The result as shown in Figure 4.Euchromosome uniform distribution in the mammary epithelial cell nuclear, abundant rough surfaced endoplasmic reticulum is arranged in the kytoplasm, arrange in groups, has rrna on it, also has abundant free ribosome, Golgi complex (Fig. 4-A1) in addition, the secretory granules of intermediate density are arranged near its mature face, and (Fig. 4-A2), cell surface have epithelial cell microvillus structure (Fig. 4-A3).The mammary gland cell of vitro culture also has apocrine feature, material aggregations such as gorky's secretory vacuole, fat granule are at the cell top, formation is rich in Microvillares top matter projection, in accumulative ribosome and some vesicles are arranged, some vesicle has medium electro-dense amorphous material.Simultaneously, and the visible distinctive cell mode of connection-desmosome of epithelial cell (Fig. 4-A4), the then formation hemidesmosome structure that has.Test-results shows that the mammary gland cell growth conditions of vitro culture is good, and the cell characteristic of secretory epithelium is complete.
Transmission electron microscope observing illustrates that to the distinctive sign of epithelial cell-desmosome structure and secretory vacuole the mammary epithelial cell of vitro culture has kept epithelial characteristics in the body.
The store method of ruminant galactophore epithelial cell of the present invention with respect to existing store method, has following advantage:
Can significantly improve freeze-stored cell vigor, help protective material and permeate to cell interior, reduce the formation of ice crystal, obtain the better protecting effect, cell long-period is preserved, perfect mammary epithelial cell lactation model.
For the advantage of the preservation method that proves ruminant galactophore epithelial cell of the present invention, the contriver has done following experiment:
Adopt ruminant galactophore epithelial cell store method of the present invention, the cow mammary gland epithelial cells of being cultivated is frozen, after preserving 2 months in the liquid nitrogen, 37 ℃ of hot water baths recovery of thawing rapidly, dye with 0.4% trypan blue, carry out viable count, the result shows that viable count accounts for 95% of the total cell count of recovery; The cell of recovery is adjusted cell concn plant in the Tissue Culture Flask 37 ℃, 5%CO again
2, cultivate under the saturated humidity condition, cell growth and form are all normal, reach the intended purposes of this experiment.
Description of drawings
Below in conjunction with accompanying drawing the specific embodiment of the present invention is described in further detail.
Fig. 1 is mammary gland cell different growing stage metamorphosis figure;
Fig. 1-A1: cultivate mammary epithelial cell upgrowth situation after 4 days; Fig. 1-A2: cultivate mammary epithelial cell upgrowth situation after 8 days; Fig. 1-A3: cultivate mammary epithelial cell upgrowth situation after 12 days; Fig. 1-A4: cultivate mammary epithelial cell upgrowth situation after 16 days; Fig. 1-A5: mammary epithelial cell excretory butterfat droplet; Fig. 1-A6: be the pebbles shape mammary epithelial cell group that paves the way; Fig. 1-A7: draw in the net the mammary epithelial cell of shape growth; Fig. 1-A8: the individual dome-like structures of mammary epithelial cell;
Fig. 2 is the mammary epithelial cell growthhabit variation diagram that goes down to posterity;
Fig. 2-A1: former generation mammary epithelial cell; Fig. 2-A2: the 4th generation mammary epithelial cell; Fig. 2-A3: the 7th generation mammary epithelial cell; Fig. 2-A4: the 9th generation mammary epithelial cell;
Fig. 3 is cow mammary gland epithelial cells CK-18 immunocytochemical stain figure as a result;
The negative control group of left figure, mammary epithelial cell is the CK-18 feminine gender; The positive experimental group of right figure, mammary epithelial cell is the CK-18 positive;
Fig. 4 is mammary epithelial cell subcellular structure figure
Fig. 4-A1: rrna on the rough surfaced endoplasmic reticulum and Golgi complex; Fig. 4-A2: intermediate density secretory granules; Fig. 4-A3: microvillus structure; Fig. 4-A4: desmosome connects.
Embodiment
In following examples:
PBS solution is: phosphate buffer soln;
EDTA: ethylenediamine tetraacetic acid (EDTA);
FBS: foetal calf serum;
DMSO: dimethyl sulfoxide (DMSO).
The separation of embodiment 1, a kind of ruminant galactophore epithelial cell, purifying and culture method, carry out following steps successively:
1), the sampling of mammary tissue:
Mammary tissue derives from the lactational holstein cow that is in that two just butchered.
In the middle of mammary gland, take a sample, separate reticular tissue and fat as far as possible, cut into 1cm earlier with 75% soaked in absolute ethyl alcohol sterilization mammary tissue, and with tissue
3The little block organization of size; Wash little block organization with 0.3% bromogeramine (being that mass concentration is 0.3% the Morpan BB aqueous solution), flushing is at least 3 times repeatedly; Again with the PBS solution that is added with bromogeramine (adding 1% Morpan BB 1ml in every 100mlPBS solution) flushing tissue, at least 3 times.
Because slaughterhouse and laboratory are not to be positioned at same place; Therefore earlier above-mentioned tissue block is immersed in 10 times of two anti-nutrient solutions (10
6The IU/L penicillin and streptomycin) in ice chest, takes back the laboratory; In super clean bench, wash repeatedly to solution then and clarify with the PBS solution that contains 1000IU/ml two anti-(penicillin and Streptomycin sulphates); In a small amount of DMEM/F12 basic culture solution (energy submergence tissue gets final product), little block organization is cut into 0.9～1.1mm
3The mammary tissue piece, with containing the two anti-basic culture solutions flushing of 100IU/ml 2-3 time, standby at last.
2), preparation nutrient solution:
Nutrient solution obtains according to following feed ratio:
In the DMEM/F12 of every ml nutrient solution, add 1 μ g hydrocortisone, 5 μ g Transferrins,iron complexess, 5 μ g Regular Insulin, 5 μ g lactotropins, 10ng Urogastron, 2 μ mol glutamine, 100IU mycillin and 0.1ml foetal calf serum; Get nutrient solution.
What do not show especially in the following step all refers to this nutrient solution.
3), tissue block is cultivated:
The mammary tissue piece of step 1) gained is put into the 50ml Tissue Culture Flask (corning) of spreading mouse tail collagen, evenly place 40-50 piece mammary tissue piece in every bottle, separate this mammary tissue piece with the sterilization toothpick, make mammary tissue piece uniform distribution, make its mutual spacing from for about 0.5cm.Put under 37 ℃, 5%CO2, saturated humidity condition and cultivate, cultivate 3～5h after; The 5ml nutrient solution is added in every then hole, puts 37 ℃, 5%CO in the incubator
2, continue under the saturated humidity condition to cultivate, the adherent property of the mammary tissue piece of inoculation is good, when being cultured to 4 days, as seen has cell to be fibroblast-like cell to outgrowth from organization edge, and launches (Fig. 1-A1) rapidly.The polygon that begins to overflow in the time of the 8th day and cobblestone-appearance epithelial cell, cell growth this moment rapidly, that cytotrophy consumes is vigorous (Fig. 1-A2).Along with epithelial effusion, fibroblastic growth begins slack-off, the fibroblast-like cells refractivity of Yi Chuing weakens at first, colour-darkening has gradually depleted sign, and epithelial cell begins a large amount of expansion (Fig. 1-A3), when density is hanged down, present polygon mostly, after density increases, the tight formation growth of cell arrangement haloing (Fig. 1-A4).(the Fig. 1-A5), illustrate that the mammary tissue of this moment still has certain secretion of visible emulsion droplet shape material around the lobule of mammary gland tissue block of cultivating.Mammary epithelial cell is flat polygon, be pebbles and pave the way that (Fig. 1-A6), close proximity between the cell show the distinctive structure (Fig. 1-A7) that draws in the net of epithelial cell to shape, after epithelial cell merges, form similar dome-shaped (dome-like) structure (Fig. 1-A8).When cell covers 80% plate bottom surface area, with mass concentration is mixture slaking liquid (the consumption 2ml/ bottle of 0.15% trypsinase and 0.02%EDTA, with D-Hanks liquid as solution) 37 ℃ of digestion above-mentioned attached cell 5-8min that suspends, digest (consumption 3ml/ bottle) with the D-Hanks liquid that contains 10%FBS (FBS mix mutually with the volume ratio of 1:9 and get) termination immediately until the retraction of 80-90% cell under inverted microscope, after becoming circle, intercellular substance and enlarging with D-Hanks; The gained cell suspension filters and the centrifugal 5min of 1000rpm/min through 105 μ m stainless steel filtering nets, with collecting cell; Then wash 3 times with nutrient solution; Adjust cell density (adopting nutrient solution to regulate) with blood cell counting plate at last, with 5 * 10
4The density of/mL is implanted culturing bottle and is cultivated, and places 37 ℃, 5%CO
2, cultivate under the saturated humidity condition; When being cultured to cell and covering 80% plate bottom surface area, carry out cell purification, method is as follows.
4), the purifying of mammary epithelial cell:
Different to the different and adherent speed of pancreatin susceptibility according to mammary epithelial cell with inoblast, adopt difference enzyme process and adherent method purifying epithelial cell repeatedly.Specific as follows:
Select above-mentioned steps 3 for use) cultivate the cell of back gained, be that the Digestive system (consumption 2ml/ bottle, with D-Hanks liquid as solution) of 0.25% pancreatin is digested to fibrocyte 10min with mass concentration earlier, remove inoblast and Digestive system; Add mass concentration again and be mixture slaking liquid (consumption 2ml/ bottle, with D-Hanks liquid as solution) the digestive epithelium cell 3-5min of 0.15% pancreatin and 0.02%EDTA; The epithelial cell that obtains waits after filtration, and counting is adjusted cell density (adopting nutrient solution to regulate) back with 5 * 10 then
4The density of/mL is planted into Tissue Culture Flask again, places 37 ℃, 5%CO
2, cultivate under the saturated humidity condition.Treat that cell spreads at the bottom of the plate at 80% o'clock and repeat successively more above-mentioned poor enzyme process and adherent method step repeatedly, the former foster mammary epithelial cell of being commissioned to train (Fig. 2-A1) after 2-3 is for the purifying that goes down to posterity, can obtain the mammary epithelial cell that prepurification is handled, and rounded paving stone sample (Fig. 2-A2).
The mammary epithelial cell of handling at above-mentioned prepurification reaches 80% and removes nutrient solution when converging; With no Ca
2+, Mg
2+Hanks liquid flushing cell, adding mass concentration then is 0.15% pancreatin and 0.02%EDTA mixture slaking liquid (consumption 2ml/ bottle, with D-Hanks liquid as solution), with peptic cell, until the retraction of 80-90% cell under inverted microscope, when becoming circle, intercellular substance expansion, stop digestion with the D-Hanks liquid of 10%FBS (3ml/ bottle, FBS mix mutually with the volume ratio of 1:9 and get) with D-Hanks; Gained be mammary epithelial cell behind the purifying;
5), the cultivation of mammary epithelial cell and growth:
With the mammary epithelial cell behind the above-mentioned purifying through the centrifugal 5min of 1000rpm/min removing Digestive system and collecting cell, then clean with nutrient solution, repeat above-mentioned steps 3 times; Remove supernatant liquor, with nutrient solution suspension cell again, and with hemocyte plate counting adjustment cell density (adopting nutrient solution to regulate), with 5 * 10
4The density of/mL is implanted culturing bottle and is cultivated, and places 37 ℃, 5%CO
2, cultivate under the saturated humidity condition, the next day change liquid.The form of passage cell has experienced initial cell island sample and has disperseed to grow, partly converge, converge to four kinds of grown forms variations of intensive growth.Nucleus is rounded or oval, and kernel is in the majority with 2-5, and cell has the contact inhibition phenomenon.In 4 generations,, substantially based on the polygon epithelial cell, with increasing of passage number, the cellular form heterogeneity presented microscler, fusiformis and trilateral mixed growth (Fig. 2-A3) with interior cell.When cell reached for the 9th generation, it is big that cell becomes, the cake sample, still visible significantly cell proliferation phenomenon (Fig. 2-A4)
The store method of embodiment 2, a kind of ruminant galactophore epithelial cell, carry out following steps successively:
1), preparation frozen solution:
Frozen solution is made up of the component of following volume percent: 10%DMSO, 40% foetal calf serum and 50% cell suspension; Cell suspension is the mixture that is formed by cell and DMEM/F12 basic medium, and the density of cell in cell suspension is 1 * 10
6/ mL, cell are the mammary epithelial cell (embodiment 1 step 5) gained) of the mammary epithelial cell (embodiment 1 step 4) gained) behind the former foster mammary epithelial cell of being commissioned to train (embodiment 1 step 3) gained), the purifying or the cultivation of going down to posterity;
2), with above-mentioned steps 1) the progressively cooling at a slow speed of the frozen solution of gained:
A, 4 ℃ reduce to-8 ℃, and rate of temperature fall is 1 ℃/min;-8 ℃ kept one minute;
B ,-8 ℃ reduce to-20 ℃, and rate of temperature fall is 0.5 ℃/min;
C ,-20 ℃ reduce to-120 ℃, and rate of temperature fall is 0.3 ℃/min;
D ,-120 ℃ frozen solution of above-mentioned steps gained is directly utilized liquid nitrogen cooling, be cooled to-196 ℃ until frozen solution;
Adopt liquid nitrogen freezing to preserve above-mentioned-196 ℃ frozen solution liquid.
Adopt aforesaid method, can guarantee that the vigor fate of mammary epithelial cell was at least 450 days.
At last, it is also to be noted that what more than enumerate only is several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Claims (3)
1. the separation of a holstein cow mammary epithelial cell, purifying culture method is characterized in that may further comprise the steps:
1), the sampling of mammary tissue:
Select the mammary gland that is separated with ruminating animal for use, the mammary gland of removing behind reticular tissue and the fat is divided into little block organization, and after sterilization and clean; Getting volume is 0.9～1.1mm
3The mammary tissue piece, standby; Described ruminating animal is a holstein cow;
2), preparation nutrient solution:
Nutrient solution obtains according to following feed ratio:
In the DMEM/F12 of every ml nutrient solution, add 1 μ g hydrocortisone, 5 μ g Transferrins,iron complexess, 5 μ g Regular Insulin, 5 μ g lactotropins, 10ng Urogastron, 2 μ mol glutamine, 100IU mycillin and 0.1ml foetal calf serum;
3), tissue block is cultivated:
The mammary tissue piece of step 1) gained is put into the cell culture container of spreading mouse tail collagen, put 37 ℃, 5%CO
2, cultivate 3～5h under the saturated humidity condition, adding nutrient solution then continues to cultivate under above-mentioned condition, when cell covered 80% container bottom surface-area, the Digestive system that with mass concentration is 0.15% trypsinase and 0.02%EDTA was in 37 ℃ of above-mentioned attached cell 5-8min of digestion suspension; After the retraction of 80-90% cell under inverted microscope, change circle and intercellular substance enlarge, stop digesting with the D-Hanks liquid that contains 10% (volume percent) FBS immediately; The cell suspension of gained gets cell after filtration with centrifugal;
Clean above-mentioned cell 3 times with nutrient solution; Adjust cell density with blood cell counting plate at last, with 5 * 10
4The density of/mL is implanted culture plate and is cultivated, and places 37 ℃, 5%CO
2, cultivate under the saturated humidity condition; When being cultured to cell and covering 80% plate bottom surface area, the former foster mammary epithelial cell of being commissioned to train;
4), the purifying of mammary epithelial cell:
With former be commissioned to train mammary epithelial cell foster and adopt difference enzyme process and adherent method repeatedly, in order to the purifying epithelial cell; Specific as follows:
Difference enzyme process: be that the Digestive system of 0.25% pancreatin is digested to fibrocyte 10min earlier, remove inoblast and Digestive system with mass concentration; Adding mass concentration again is the mixture slaking liquid digestive epithelium cell 3-5min of 0.15% pancreatin and 0.02%EDTA;
Adherent method repeatedly: with the epithelial cell that obtains after filtration, after counting adjusts cell density, with 5 * 10
4The density of/mL is planted into Tissue Culture Flask again, places 37 ℃, 5%CO
2, cultivate under the saturated humidity condition;
Treat that cell spreads at the bottom of the plate at 80% o'clock, repeat above-mentioned poor enzyme process and adherent method 1～2 time repeatedly more successively, obtain the mammary epithelial cell after prepurification is handled;
Mammary epithelial cell after above-mentioned prepurification is handled reaches 80% when converging, and removes nutrient solution; With no Ca
2+, Mg
2+Hanks liquid flushing cell, adding mass concentration then is 0.15% pancreatin and 0.02%EDTA mixture slaking liquid, with peptic cell, until the retraction of 80-90% cell under inverted microscope, when becoming circle and intercellular substance expansion, with the D-Hanks liquid termination digestion of 10% (volume percent) FBS; Gained be mammary epithelial cell behind the purifying;
5), the cultivation of going down to posterity of mammary epithelial cell:
Mammary epithelial cell behind the above-mentioned purifying through centrifugal, is got cell, then clean cell 3 times with nutrient solution; Remove supernatant liquor, with nutrient solution suspension cell again, and with hemocyte plate counting adjustment cell density, with 5 * 10
4The density of/mL is implanted culturing bottle and is cultivated, and places 37 ℃, 5%CO
2, cultivate under the saturated humidity condition, the next day change liquid.
2. the separation of holstein cow mammary epithelial cell according to claim 1, purifying culture method is characterized in that: in the described step 3), the distance of spreading between the mammary tissue piece in the cell culture container of mouse tail collagen is 0.45～0.55cm.
3. the separation of holstein cow mammary epithelial cell according to claim 2, purifying culture method is characterized in that: in the described step 3), cell suspension is the stainless steel filtering net filtration of 105 μ m with the aperture.
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| CN102140433B (en) * | 2010-12-28 | 2012-12-26 | 北京民海生物科技有限公司 | Cell freezing method |
| CN102648708B (en) * | 2011-02-25 | 2014-08-13 | 深圳华大方舟生物技术有限公司 | Freezing liquid for embryo or cells and application thereof |
| CN103103159A (en) * | 2013-02-18 | 2013-05-15 | 天津农学院 | Cow mammary gland epithelial cell nutrient solution |
| CN103289950A (en) * | 2013-06-28 | 2013-09-11 | 浙江大学 | Methods for in vitro separation, culture, preservation and recovery of milk cow omasum epithelial cells |
| CN104195100A (en) * | 2014-08-29 | 2014-12-10 | 山东省农业科学院畜牧兽医研究所 | In-vitro culture method of mammary gland epithelial cells |
| CN105010307A (en) * | 2015-07-08 | 2015-11-04 | 中国检验检疫科学研究院 | Cryopreservation solution and cryopreservation resuscitation method for liver primary cells |
| CN106190952A (en) * | 2016-07-19 | 2016-12-07 | 安徽惠恩生物科技股份有限公司 | A kind of amplifying preparation process of epithelial cell |
| CN115074315B (en) * | 2022-06-08 | 2023-10-31 | 四川省畜牧科学研究院 | High-quality pig ear tissue sampling and long-term high-efficiency preservation method |
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