CN101861857B - Preservation method of ruminant mammary gland epithelial cells - Google Patents
Preservation method of ruminant mammary gland epithelial cells Download PDFInfo
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Abstract
The invention discloses a preservation method of ruminant mammary gland epithelial cells. The preservation method comprises the following steps of: (1) preparing a cryoprotective solution which is prepared from the following components in percentage by weight: 10 percent of DMSO (Dimethyl Sulfoxide), 40 percent of fetal calf serums and 50 percent of cell suspension; and (2) gradually and slowly lowering the temperature of the obtained cryoprotective solution until the temperature of the cryoprotective solution is lowered to -196 DEG C and cryopreserving the cryoprotective solution at -196 DEG C with liquid nitrogen. By adopting the method of the invention, the preservation time of the ruminant mammary gland epithelial cells can be prolonged.
Description
The present invention is to be that 2008.11.24, application number are dividing an application of 200810162422.3 " the separation and purification method and the store methods of ruminant galactophore epithelial cell " applying date.
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 galactophore epithelial cell.
Background technology
Mammary gland is important skin exocrine secretion organ, has multiple physiology, biochemistry and immunologic function, can prescribing adequate nutrition 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 galactophore epithelial cell, function differentiation, 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, alimentology, 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 culture in vitro of complicacy, can well solve problem for specific factor research.Successively there is the scholar that the culture in vitro 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 glandular cell, and for separating 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, microstructure is observed, Study of Cryopreservation of Human is few, to the system research of its lactation function still less.
Summary of the invention
The technical problem that the present invention will solve 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 holding time.
In order to solve the problems of the technologies described above, the present invention provides a kind of separation, purifying cultivation of ruminant galactophore epithelial cell, may further comprise the steps:
1), the sampling of breast tissue:
Select the mammary gland that is separated with ruminant for use, the mammary gland of removing behind connective tissue and the fat is divided into little block organization, and after sterilization and clean; Getting volume is 0.9～1.1mm
3The breast tissue piece, subsequent use;
2), preparation culture fluid:
Culture fluid obtains according to following rate of charge:
In the DMEM/F12 of every ml culture fluid, add 1 μ g hydrocortisone, 5 μ g transferrins, 5 μ g insulin, 5 μ g prolactin(PRLs, 10ng EGF, 2 μ mol glutamine, 100IU mycillin and 0.1ml hyclone;
3), piece of tissue is cultivated:
The breast 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, using mass concentration was that the digestive juice of 0.15% trypsase and 0.02%EDTA is 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 space between cells enlarge, stop digesting with the D-Hanks liquid that contains 10% (percent by volume) FBS immediately; The cell suspension of gained gets cell through filtration and centrifugal;
Clean above-mentioned cell 3 times with culture fluid; Adjust cell density (adopting culture fluid 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 galactophore epithelial cell of being commissioned to train;
4), the purifying of galactophore epithelial cell:
With former be commissioned to train galactophore epithelial cell foster and adopt difference enzyme process and adherent method repeatedly, in order to the purifying epithelial cell; Specific as follows:
The difference enzyme process: elder generation's use mass concentration is that the digestive juice of 0.25% pancreatin is digested to fibrocyte 10min, removes fibroblast and digestive juice; 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 through filter with centrifugal, counting adjustment cell density (the employing culture fluid dilutes) after, 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 above-mentioned poor enzyme process and adherent method 1～2 time repeatedly more successively, obtain the galactophore epithelial cell after prepurification is handled;
Galactophore epithelial cell after above-mentioned prepurification is handled reaches 80% when converging, and removes culture fluid; With no Ca
2+, Mg
2+Hanks liquid cells washed; Adding mass concentration then is 0.15% pancreatin and 0.02%EDTA mixture slaking liquid; With vitellophag, until the retraction of 80-90% cell under inverted microscope, when becoming circle and space between cells expansion, with the D-Hanks liquid termination digestion of 10%FBS; Gained be the galactophore epithelial cell behind the purifying;
5), the cultivation of going down to posterity of galactophore epithelial cell:
Galactophore epithelial cell behind the above-mentioned purifying through centrifugal, is got cell, then clean cell 3 times with culture fluid; Remove supernatant, with culture fluid suspension cell again, and with haemocyte plate counting adjustment cell density (adopting culture fluid to dilute), with 5 * 10
4The density of/mL is implanted blake 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 cultivation: in the step 3), the distance of spreading between the breast tissue piece in the cell culture container of mouse tail collagen is 0.45～0.55cm; It is the stainless steel filtering net filtration of 105 μ m that cell suspension uses 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 percent by volume: 10%DMSO, 40% hyclone and 50% cell suspension; Said cell suspension is the mixture that is formed by cell and DMEM/F12 basal medium, and the density of cell in cell suspension is 1 * 10
6/ mL, said cell are the former galactophore epithelial cell of being commissioned to train foster galactophore epithelial cell, the galactophore 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 frozen to preserve above-mentioned-196 ℃ frozen solution.
The present invention is intended to set up the galactophore 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, disease prevention and cure; Processes such as galactophore 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% trypsinization fibroblast; 0.15% pancreatin and 0.02%EDTA digestive epithelium cell) and repeatedly the adherent method purifying epithelial cell, find that in the DMEM/F-12 culture fluid that has added compositions such as insulin, prolactin(PRL, transferrins, cell is typical epithelium appearance 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 inventor has made following confirmatory experiment:
Experiment 1, inventor galactophore epithelial cell and the galactophore epithelial cell of cultivating that goes down to posterity of step 5) gained after to the purifying of step 4) gained carried out following evaluation, and be 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 glandular cell.
The result is as shown in Figure 3, and find: the CK-18 specificity is expressed in galactophore epithelial cell, and has detected identical result in the epithelial cell after going down to posterity, and explains that the mammary glandular cell keratin characteristic (epithelium characteristic) of cultivating still exists.
Collection is gone down to posterity and is cultured to the 9th generation eugonic mammary glandular cell (1-5 * 10
6Individual); The centrifugal 10min of 1000r/min removes supernatant, with 4 ℃ of fixed overnight of cold 2.5% glutaraldehyde; Through 0.1M phosphate buffer rinsing sample three times; Fix with 1% osmic acid again,, under transmission electron microscope, observe the ultra microstructure of mammary glandular cell after steps such as the dehydration of ethanol gradient, embedding, polymerization, section, dyeing are processed ultra-thin section.The result is as shown in Figure 4.Autosome evenly distributes in the galactophore epithelial cell nuclear; Abundant rough surfaced endoplasmic reticulum (RER) is arranged in the kytoplasm; Arrange in groups, with ribosome, also have the free ribosome that enriches, Golgi complex (Fig. 4-A1) in addition it on; 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 glandular cell of culture in vitro also has apocrine characteristic; Material aggregations such as Gorky's secretory vacuole, fat drop are at the cell top; Formation is rich in Microvillares top matter projection, in gathering arranged ribosome and some vesicles, some vesicle has medium electro-dense amorphous material.Simultaneously, and the visible distinctive cell connected mode-desmosome of epithelial cell (Fig. 4-A4), the then formation hemidesmosome structure that has.Result of the test shows that the mammary glandular cell growth conditions of culture in vitro is good, and the cell characteristic of secretory epithelium is complete.
Transmission electron microscope observing explains that to the distinctive sign of epithelial cell-desmosome structure and secretory vacuole the galactophore epithelial cell of culture in vitro 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 protectant and permeate to cell interior, reduce the formation of ice crystal, obtain the better protect effect, cell long-period is preserved, perfect galactophore epithelial cell lactation model.
For the advantage of the preservation method that proves ruminant galactophore epithelial cell of the present invention, the inventor 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 baths recovery of thawing is rapidly dyeed with 0.4% trypan blue; Carry out viable count, the result shows that viable count accounts for 95% of the total cell number of recovery; The cell of recovery is adjusted cell concentration 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
Do further explain below in conjunction with the accompanying drawing specific embodiments of the invention.
Fig. 1 is mammary glandular cell different growing stage metamorphosis figure;
Fig. 1-A1: cultivate galactophore epithelial cell upgrowth situation after 4 days; Fig. 1-A2: cultivate galactophore epithelial cell upgrowth situation after 8 days; Fig. 1-A3: cultivate galactophore epithelial cell upgrowth situation after 12 days; Fig. 1-A4: cultivate galactophore epithelial cell upgrowth situation after 16 days; Fig. 1-A5: the butterfat droplet of galactophore epithelial cell secretion; Fig. 1-A6: be the cobblestone shape galactophore epithelial cell crowd that paves the way; Fig. 1-A7: draw in the net the galactophore epithelial cell of shape growth; Fig. 1-A8: the individual dome-like structures of galactophore epithelial cell;
Fig. 2 is the galactophore epithelial cell growthform variation diagram that goes down to posterity;
Fig. 2-A1: former generation galactophore epithelial cell; Fig. 2-A2: the 4th generation galactophore epithelial cell; Fig. 2-A3: the 7th generation galactophore epithelial cell; Fig. 2-A4: the 9th generation galactophore 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, galactophore epithelial cell is the CK-18 feminine gender; The positive experimental group of right figure, galactophore epithelial cell is the CK-18 positive;
Fig. 4 is galactophore epithelial cell subcellular structure figure
Fig. 4-A1: ribosome on the rough surfaced endoplasmic reticulum (RER) 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: PBS;
EDTA: ethylenediamine tetra-acetic acid;
FBS: hyclone;
DMSO: dimethyl sulfoxide (DMSO).
The separation of embodiment 1, a kind of ruminant galactophore epithelial cell, purifying and cultivation, carry out following steps successively:
1), the sampling of breast tissue:
Breast tissue derives from the lactational holstein cow that is in that two just butchered.
In the middle of mammary gland, take a sample, separate connective tissue and fat as far as possible, cut into 1cm earlier with 75% soaked in absolute ethyl alcohol sterilization breast 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 benzalkonium bromide aqueous solution), flushing is at least 3 times repeatedly; Again with the PBS solution that is added with bromogeramine (adding 1% benzalkonium bromide 1ml in every 100mlPBS solution) washed tissue, at least 3 times.
Because slaughterhouse and laboratory are not to be positioned at same place; Therefore earlier above-mentioned piece of tissue is immersed in 10 times of two anti-culture fluids (106IU/L is blue or green, streptomycin) and 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 streptomycins); In a small amount of DMEM/F12 basic culture solution (ability submergence tissue gets final product), little block organization is cut into 0.9～1.1mm
3The breast tissue piece, with containing the two anti-basic culture solutions flushing of 100IU/ml 2-3 time, subsequent use at last.
2), preparation culture fluid:
Culture fluid obtains according to following rate of charge:
In the DMEM/F12 of every ml culture fluid, add 1 μ g hydrocortisone, 5 μ g transferrins, 5 μ g insulin, 5 μ g prolactin(PRLs, 10ng EGF, 2 μ mol glutamine, 100IU mycillin and 0.1ml hyclone; Get culture fluid.
What do not show especially in the following step all refers to this culture fluid.
3), piece of tissue is cultivated:
The breast 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 breast tissue piece in every bottle; Separate this breast tissue piece with the sterilization toothpick, the breast tissue piece is evenly distributed, its mutual spacing is left for about 0.5cm.Put 37 ℃, 5%CO
2, cultivate under the saturated humidity condition, cultivate 3～5h after; The 5ml culture fluid 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 breast tissue piece of inoculation is good, when being cultured to 4 days, visible have cell to be fibroblast-like cell to outgrowth from organization edge, and launch (Fig. 1-A1) rapidly.The polygonal 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, and the fibroblast-like cells refractivity of overflowing at first weakens; Colour-darkening has gradually depleted sign, and epithelial cell begins a large amount of expansion (Fig. 1-A3); When density is hanged down; Present polygonal mostly, after density increases, the tight formation growth of cell arrangement haloing (Fig. 1-A4).(the Fig. 1-A5), explain that the breast tissue of this moment still has certain secreted of visible emulsion droplet shape material around the lobule of mammary gland piece of tissue of cultivating.Galactophore epithelial cell is flat polygonal; Be cobblestone 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; Using mass concentration is mixture slaking liquid (the consumption 2ml/ bottle of 0.15% trypsase 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 1: 9 volume ratio and get) termination immediately until the retraction of 80-90% cell under inverted microscope, after becoming circle, space between cells 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 culture fluid; Adjust cell density (adopting culture fluid to regulate) with blood cell counting plate at last, with 5 * 10
4The density of/mL is implanted blake 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 following.
4), the purifying of galactophore epithelial cell:
Different to the different and adherent speed of pancreatin susceptibility according to galactophore epithelial cell with fibroblast, 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, using mass concentration earlier is that the digestive juice (consumption 2ml/ bottle, with D-Hanks liquid as solution) of 0.25% pancreatin is digested to fibrocyte 10min, removes fibroblast and digestive juice; 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 is through filtering etc., and counting adjustment cell density (adopting culture fluid to regulate) back is 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 galactophore 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 galactophore epithelial cell that prepurification is handled, and rounded paving stone appearance (Fig. 2-A2).
The galactophore epithelial cell of handling at above-mentioned prepurification reaches 80% and removes culture fluid when converging; With no Ca
2+, Mg
2+Hanks liquid cells washed; 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 vitellophag, until 80-90% cell under inverted microscope retraction, become circle, when the space between cells enlarges; Stop digestion with the D-Hanks liquid of 10%FBS (3ml/ bottle, FBS mix mutually with 1: 9 volume ratio and get) with D-Hanks; Gained be the galactophore epithelial cell behind the purifying;
5), the cultivation of galactophore epithelial cell and growth:
With the galactophore epithelial cell behind the above-mentioned purifying through the centrifugal 5min of 1000rpm/min removing digestive juice and collecting cell, then clean with culture fluid, repeat above-mentioned steps 3 times; Remove supernatant, with culture fluid suspension cell again, and with haemocyte plate counting adjustment cell density (adopting culture fluid to regulate), with 5 * 10
4The density of/mL is implanted blake 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 appearance and has disperseed to grow, partly converge, converge to four kinds of grown forms variations of intensive growth.Cell nucleus is rounded or oval, and kernel is in the majority with 2-5, and cell has the contact inhibition phenomenon.4 generations were main with the polygonal epithelial cell with interior cell basically, and with increasing of passage number, the cellular morphology heterogeneity presents microscler, fusiformis and triangle mixed growth (Fig. 2-A3).When cell reached for the 9th generation, it is big that cell becomes, cake appearance, 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 percent by volume: 10%DMSO, 40% hyclone and 50% cell suspension; Cell suspension is the mixture that is formed by cell and DMEM/F12 basal medium, and the density of cell in cell suspension is 1 * 10
6/ mL, cell are the galactophore epithelial cell (embodiment 1 step 5) gained) of the galactophore epithelial cell (embodiment 1 step 4) gained) behind the former foster galactophore 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 frozen to preserve above-mentioned-196 ℃ frozen solution liquid.
Adopt said method, can guarantee that the vigor fate of galactophore 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 (1)
1. the store method of a ruminant galactophore epithelial cell is characterized in that may further comprise the steps:
1), preparation frozen solution:
Frozen solution is made up of the component of following percent by volume: 10%DMSO, 40% hyclone and 50% cell suspension;
Said cell suspension is the mixture that is formed by cell and DMEM/F12 basal medium, and the density of cell in cell suspension is 1 * 10
6/ mL, said cell are the former galactophore epithelial cell of being commissioned to train foster galactophore epithelial cell, the galactophore epithelial cell behind the purifying or going down to posterity and cultivating of ruminant; Said ruminant is for being in lactational holstein cow;
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 frozen to preserve above-mentioned-196 ℃ frozen solution.
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| 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 |
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| CN101302494A (en) * | 2008-06-23 | 2008-11-12 | 中国科学院东北地理与农业生态研究所 | Milk cow mammary glandular cell in vitro isolated culture method |
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| 多曙光等.牛乳腺上皮细胞的分离培养及其生物学特性.《动物学研究》.2006,第27卷(第3期),299-305. * |
| 王亨等.荷斯坦奶牛乳腺上皮细胞的体外培养.《畜牧与兽医》.2007,第39卷(第4期),41-43. * |
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