CN104488852A - Frozen stock solution for storing mammary epithelial cells of milk goat and freeze-saving method - Google Patents
Frozen stock solution for storing mammary epithelial cells of milk goat and freeze-saving method Download PDFInfo
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- CN104488852A CN104488852A CN201410814668.XA CN201410814668A CN104488852A CN 104488852 A CN104488852 A CN 104488852A CN 201410814668 A CN201410814668 A CN 201410814668A CN 104488852 A CN104488852 A CN 104488852A
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Abstract
The invention discloses a frozen stock solution for storing mammary epithelial cells of a milk goat. The frozen stock solution is prepared by mixing a basic frozen stock solution and insulin-transferrin-sodium selenate in a volume ratio of 100 to (1-2). The basic frozen stock solution is prepared by mixing 10% by volume percent of dimethyl sulfoxide, 30-50% by volume percent of a DMEM/F12 culture medium and 40-60% by volume percent of fetal calf serum. The freeze-saving frozen stock solution disclosed by the invention is few in step, simplified in process and low in cost, the resuscitated mammary epithelial cells are strong in anti-apoptosis performance, oxidization resistance and multiplication capacity, the resuscitation rate of the mammary epithelial cells can be increased, and the vitality of the resuscitated cells reaches up to above 95%, so that the cell growth quality is ensured, the proliferation cycle of cell resuscitation is shortened and the mammary epithelial cells of the milk goat can be saved for a long time.
Description
Technical field:
The present invention relates to tissue and cell engineering, technical field of cell biology, be specifically related to a kind of preservation epithelioglandular cryopreserving liquid of Goats Milk and cryopreservation methods.
Background technology
Goats Milk glandular epithelium not only can be expressed as research milk protein gene, apoptotic model, can also as the detection system of mammary gland specific expression vector.At present, though Goats Milk glandular epithelium In-vitro separation culture method is set up substantially, but the Goats Milk galandular epithelium immortal cell line of also not generally acknowledging.Goats Milk glandular epithelium is the cell of original cuiture, and need through the purifying of cell and Secondary Culture to obtain functional galactophore epithelial cell, not only cultivation cycle is long, but also causes the waste of a large amount of reagent, material; And the life-span of passage cell is also limited, along with cell chulture algebraically increases, ability of cell proliferation can decline, and also can impact result of the test.Therefore, frozen realization must be utilized the long-term preservation of the galactophore epithelial cell obtained, for laboratory research work provides convenient, meet the research needs utilizing galactophore epithelial cell.
Although successively have scholar to be studied the epithelioglandular culture in vitro of Goats Milk both at home and abroad, but research mainly at cultural method, morphology and hormone in the regulation and control of mammary glandular cell etc., research for freezen protective is also few, mainly still utilize traditional store method, and the cell cryopreservation condition that people use is inconsistent, cell viability after cell recovery is also all lower, generally all lower than 75%, makes Goats Milk glandular epithelium cannot realize long-term preservation.Chinese patent CN101861857A discloses the store method of ruminant galactophore epithelial cell; cryopreserving liquid is DMSO, 40% hyclone and 50% cell suspension of 10%; adopt progressively at a slow speed falling temperature method carry out frozen;-8 DEG C ,-20 DEG C ,-120 DEG C are down to successively by 4 DEG C; again the frozen solution of-120 DEG C is directly utilized liquid nitrogen cooling, adopt liquid nitrogen frozen to preserve until be cooled to-196 DEG C.This patent only illustrates that the vigor number of days of galactophore epithelial cell is at least 450 days, but does not study cell viability, form, proliferative conditions etc. after cell recovery rate and recovery.Said method, protect composition single in cells frozen storing liquid, step is more, and interval time is long, and the carelessness of each step can have a strong impact on the survival of cell.
Insulin-transferrins-selenium sodium additives comprises insulin, transferrins and selenium 3 kinds of compositions.Wherein insulin is a kind of proteohormone, and by islet β cell, have the effect promoting the synthesis of glycogen, fat and protein, galactophore epithelial cell can produce and excreting insulin associated proteins, by being combined the propagation promoting cell with insulin.Transferrins can promote epithelial mitosis, in conjunction with iron ion, can also reduce the utilization of its toxicity and promotion cell.Selenium element is a kind of desirable antioxidant, can form antioxidase, and energy Cell protection film, from oxidative damage, keeps its permeability.
Summary of the invention
The technical problem to be solved in the present invention is to provide that a kind of step is few, technique simplifies, cost is low, galactophore epithelial cell recovery after anti-apoptotic, anti-oxidant and multiplication capacity is strong, the anabiosis rate of frozen galactophore epithelial cell can be improved, after recovery, the vigor of cell is up to more than 95%, ensure Growth of Cells quality, shorten the proliferating cycle of cell recovery, the epithelioglandular cryopreserving liquid of Goats Milk and cryopreservation methods can be preserved for a long time.
The present invention solves the problems of the technologies described above by the following technical solutions:
The epithelioglandular cryopreserving liquid of a kind of preservation Goats Milk, is mixed 100:1-2 by volume by basic cryopreserving liquid and insulin-transferrins-selenium sodium;
Described basic cryopreserving liquid by percent by volume be the dimethyl sulfoxide (DMSO) of 10%, the DMEM/F12 medium of 30-50% and 40 – 60% hyclones mix, above-mentioned three components percent by volume summation is 100%.
Use the cryopreservation methods of the epithelioglandular cryopreserving liquid of preservation Goats Milk described in claim 1, comprise the following steps:
(1), the Goats Milk glandular epithelium in vegetative period of taking the logarithm, adding mass concentration is the trypsase of 0.25% and the EDTA mixed solution of 0.02%, is placed in 37 DEG C, 5%C0
2constant incubator in digest, until cytoplasm retraction, space between cells increase, cell shorten circle into time, with containing percent by volume 10% hyclone termination medium stop digestion, centrifugal, supernatant discarded, obtains Goats Milk glandular epithelium;
(2), gained Goats Milk glandular epithelium adds described cryopreserving liquid, and piping and druming evenly, forms cell suspension, cell suspension is placed in cryopreservation tube;
(3), by above-mentioned cryopreservation tube be closed in foam freezing storing box, foam freezing storing box is placed 15h under-80 DEG C of cryogenic conditions;
(4), preserve in foam freezing storing box immersion liquid nitrogen.
In described step (1), centrifugal rotational speed is 1500rpm, centrifugation time 10min.
In described step (2), the cell concentration of cell suspension is 0.5-1 × 10
7individual/mL.
The upper surface of described foam freezing storing box is porose, and the degree of depth in hole is greater than the height of described cryopreservation tube, and described cryopreservation tube inserts in the hole, and aperture foam block is closed.
Marked improvement of the present invention has:
1, compared with the prior art, eliminate step, simplify technique, have compressed cost;
2, cryopreservation tube is closed in foam freezing storing box by cryopreservation methods of the present invention, cryopreservation tube can be avoided directly to contact with low temperature environment, cryopreservation tube inner cell can be made again to lower the temperature rapidly, ensures that the vigor of the rear cell of recovery is up to more than 95%.
3, the cells frozen storing liquid containing insulin-transferrins-selenium sodium is adopted, anti-apoptotic, the oxidation resistance of Goats Milk glandular epithelium anti-freezing injury ability and the rear cell of recovery can be improved, after recovery, the vigor of cell is high, form is normal, refractivity good, flush, doubling time is short, and growth curve meets the general biological regularity of Growth of Cells; A large amount of keratin 5s and the positive particle of CK8 is had in recovery cell cytosol, in galactophore epithelial cell, the expression of beta-casein gene is 10 times in conventional cryopreservation cell, ensure that the epithelioglandular biological property of Goats Milk is constant, promote the milk protein secreting function keeping recovery cell; Realize the vigor of the rear cell of recovery up to more than 95%, and shorten the proliferating cycle of cell recovery, ensure that Growth of Cells quality, cell can be preserved for a long time.
4, adopt this method, the vigor number of days of galactophore epithelial cell is at least 700 days.
Accompanying drawing explanation
Fig. 1 be the embodiment of the present invention 1 cryopreserving liquid frozen galactophore epithelial cell recovery after cellular morphology figure, multiplication factor is 200 times;
Fig. 2 be contrast experiment example cryopreserving liquid frozen galactophore epithelial cell recovery after cellular morphology figure, multiplication factor is 200 times;
Fig. 3 be the embodiment of the present invention 1-3 and contrast experiment example cryopreserving liquid frozen galactophore epithelial cell recovery growth curve chart; Wherein, curve A 1, A2, A3, A4 are respectively and embodiment 1, embodiment 2, embodiment 3, the routine corresponding growth curve of contrast experiment;
Fig. 4 is the recovery cell division phasor of the frozen galactophore epithelial cell of the embodiment of the present invention 1 cryopreserving liquid; Observation condition is oily mirror, multiplication factor is 1000 times;
Fig. 5 is the recovery cytokeratin SABC qualification figure of the frozen galactophore epithelial cell of the embodiment of the present invention 1 cryopreserving liquid;
Fig. 6 expresses negative control figure as the Goat Fibroblasts keratin of Fig. 5.
Embodiment
Elaborate below in conjunction with Fig. 1-6 pairs of the specific embodiment of the present invention, but do not form limiting the scope of the invention.
Embodiment 1:
The epithelioglandular cryopreserving liquid of a kind of preservation Goats Milk, be diluted in the basic cryopreserving liquid of 100mL by the insulin-transferrins-selenium sodium of 1mL, described basic cryopreserving liquid is mixed by following component:
DMEM/F12 culture fluid 30mL;
Hyclone 60mL;
Dimethyl sulfoxide (DMSO) 10mL.
Embodiment 2:
The epithelioglandular cryopreserving liquid of a kind of preservation Goats Milk, be diluted in the basic cryopreserving liquid of 100mL by the insulin-transferrins-selenium sodium of 1.5mL, described basic cryopreserving liquid is mixed by following component:
DMEM/F12 culture fluid 40mL;
Hyclone 50mL;
Dimethyl sulfoxide (DMSO) 10mL.
Embodiment 3:
The epithelioglandular cryopreserving liquid of a kind of preservation Goats Milk, be diluted in the basic cryopreserving liquid of 100mL by the insulin-transferrins-selenium sodium of 2mL, described basic cryopreserving liquid is mixed by following component:
DMEM/F12 culture fluid 50mL;
Hyclone 40mL;
Dimethyl sulfoxide (DMSO) 10mL.
Contrast experiment's example:
Common cryopreserving liquid, be also cryopreserving liquid conventional at present, concrete component is:
DMEM/F12 culture fluid 50mL
Hyclone 40mL
Dimethyl sulfoxide (DMSO) 10mL
(1), use the frozen Goats Milk glandular epithelium of cryopreserving liquid of embodiment 1-3 and contrast experiment's example respectively, cryopreservation methods comprises the following steps:
(1), the cell in vegetative period of taking the logarithm, with mass concentration 0.25%Trypsin and mass concentration 0.02%EDTA mixed solution in 37 DEG C, 5%C0
2constant incubator in digest Goats Milk glandular epithelium, when cytoplasm retraction, space between cells increase, cell shorten circle into, digestion is stopped with the termination culture fluid containing percent by volume 10% hyclone, proceed in the centrifuge tube of 10mL centrifugal, centrifugal rotational speed 1500rpm, centrifugation time 10min, supernatant discarded;
(2), add above-mentioned homemade cells frozen storing liquid, and piping and druming evenly, formation cell concentration is 0.5-1 × 10
7the cell suspension of individual/mL, installs in cryopreservation tube by cell suspension by every pipe 1.5mL;
(3), by cryopreservation tube be closed in foam freezing storing box, foam freezing storing box be placed in-80 DEG C of low temperature refrigerators and place 15h;
(4), preserve in foam freezing storing box immersion liquid nitrogen.
The foam cube block of foam freezing storing box to be the length of side be 10cm, thereon surface uniform make a call to 3 holes, hole depth is 8cm, by be highly in 5cm cryopreservation tube patchhole after, use foam piece closed orifices again, cryopreservation tube is closed in the middle of foam box, avoid cryopreservation tube directly to contact with low temperature environment.
(2) above-mentioned frozen cell recovery method:
(1), from liquid nitrogen take out cryopreservation tube, in the water-bath of 37 DEG C, be incubated 1-2min, make it melt completely;
(2), with cell culture fluid dilute cryopreserving liquid, proceed in the centrifuge tube of 10mL centrifugal, centrifugal rotational speed 1500rpm, centrifugation time 10min, supernatant discarded;
(3), add cell culture fluid formation cell suspension, and the concentration of cell suspension is adjusted to 5 × 10
5individual/ml, puts 37 DEG C, 5%C0
2constant incubator in cultivate.
(3) above-mentioned frozen cell recovery effect detection:
1, cell recovery rate is detected
Get the cell suspension after cell recovery, add 0.1% trypan blue solution of 10 μ L by every mL cell suspension, mix, leave standstill 10min.Draw mixed liquor, in instillation cell counting count board, examine under a microscope counting.All refractivities by force and not tinter are living cells, and contaminate blue person for dead cell, count 1000 cells, calculate cell viability, concrete formula is:
Living cell rate (%)=total viable cell/(total viable cell+dead cell sum) × 100;
Resuscitation effect shows, the galactophore epithelial cell anabiosis rate adopting the cryopreserving liquid of embodiment 1 frozen is 96%, the galactophore epithelial cell anabiosis rate adopting the cryopreserving liquid of embodiment 2 frozen is 96%, the galactophore epithelial cell anabiosis rate adopting the cryopreserving liquid of embodiment 3 frozen is 98%, and the galactophore epithelial cell anabiosis rate adopting the cryopreserving liquid of contrast experiment's example frozen is 70%.
2, the morphological observation after cell recovery
Adopt the cell that the cryopreserving liquid of embodiment 1,2,3 is frozen, in cell recovery process, cultivate 2h galactophore epithelial cell and start adherent, under inverted microscope, observe the cell state after recovery, still keep several characteristic to tens cell aggregation growths in process of growth, cellular morphology is good; After cultivating 24h, the galactophore epithelial cell large area adherent growth of visible more than 95%, without suspension cell, cell is the growth of paving stone sample, and size is even, and refractivity is good, exhibits vigour vigorous, as shown in Figure 1.Adopt the cryopreserving liquid of contrast experiment's example, after 24h is cultivated in recovery, suspension cell is more, and the cell shape of adherent growth is irregular, and refractivity is poor, and vigor is lower, as shown in Figure 2.
3, the biological character of recovery cell detects:
(1) mensuration of recovery cell doubling time
By the galactophore epithelial cell after recovery with 5 × 10
4individual/mL is inoculated in six well culture plates, at 37 DEG C, 5%CO
2, saturated humidity cultivates, after inoculation, 120h vitellophag also counts.Specific formula for calculation is:
T﹦tlog2/(logNt/N0)
Wherein, T is the cell population doublings time, and t is incubation time (h), and N0 is primary vaccination cell number, and Nt is the cell number of t time.
Measurement result is as shown in table 1:
Table 1 Goat Mammary Epithelial Cells recover in different cryopreserving liquid after population doubling time
In table 1, capitalization A, B represent that difference extremely significantly (P<0.01).
Table 1 result illustrates, adopt the cryopreserving liquid of embodiment 1,2,3, the population doubling time of recovery cell is shorter; And adopting the cryopreserving liquid of contrast experiment's example, population doubling time is then longer; Illustrate adopt cryopreserving liquid of the present invention and cryopreservation methods ability of cell proliferation stronger.
(2) mensuration of recovery cell growth curve
By the galactophore epithelial cell after recovery with 1 × 10
4the density of individual/mL is inoculated in 24 porocyte culture plates, in 37 DEG C and 5%CO
2, cultivate in the incubator of saturated humidity.The every day same time digests 3 porocytes with TE digestive juice, after blood counting chamber counts respectively, averages.Continuous 8 days.Take incubation time as abscissa, the mean of 3 porocytes is that ordinate draws cell growth curve, as shown in Figure 3.Growth curve shows: Goat Mammary Epithelial Cells growth 0-2d demurrage, exponential phase 2-6d, 7d enters plateau, illustrate: the Goat Mammary Epithelial Cells growth curve of three embodiments is all S-type, all meet the general biological regularity of Growth of Cells, there is normal division growth characteristic, but the cell division multiplication capacity of contrast experiment's example is poor.
(3) karyotyping
The galactophore epithelial cell of recovery is added colchicine 0.1 μ g/mL at exponential growth animated period continue to cultivate 1h, digest with TE digestive juice, 1200rpm centrifugal cell harvesting, with the pre-warm hypotonic 15 ~ 20min of KCl solution to 37 DEG C of 0.075mol/L, after add Fresh fixative 0.5 ~ 1mL and pre-fix, fixer is by methyl alcohol: acetic acid mixes by volume at 3: 1; Then the centrifugal 8 ~ 10min of 1200rpm, abandoning supernatant; Repeat to fix 3 times with 5 ~ 8mL fixer, fix 20min at every turn, then the centrifugal 8 ~ 10min of 1200rpm; Make cell suspension with a small amount of Fresh fixative, drip 2 ~ 3 cell suspensions on the clean slide of precooling, cold wind dries up, Giemsa dyeing, washing, dry, microscopy.Sprawl intact mid-term to 30 under the microscope and add up chromosome number mutually, represent this cell line cell chromosome number with the maximum person of the cell number that chromosome number is identical.
Statistics: the chromosome number distribution of Goat Mammary Epithelial Cells system is 56-120, wherein:
Embodiment 1, chromosome number be 60 cell account for 92%, chromosome number be 56 cell account for 5%, chromosome number is that other cell accounts for 3%.Result shows that the cell of 92% has normal chromosome number (2n=60); Be illustrated in figure 4 the split coil method of embodiment 1.
Embodiment 2, chromosome number be 60 cell account for 90%, chromosome number be 56 cell account for 3%, chromosome number is that other cell accounts for 7%.Result shows that the cell of 90% has normal chromosome number (2n=60);
Embodiment 3, chromosome number be 60 cell account for 94%, chromosome number be 56 cell account for 2%, chromosome number is that other cell accounts for 4%.Result shows that the cell of 94% has normal chromosome number (2n=60);
Contrast experiment example, chromosome number be 60 cell account for 83%, chromosome number be 56 cell account for 12%, chromosome number is that other cell accounts for 5%.Result shows that the cell of 83% has normal chromosome number (2n=60).
Above-mentionedly show, three embodiments and contrast experiment's example, the cell after recovery is more stable, and cell transformation does not occur, but three embodiments are all than more stable to the galactophore epithelial cell more frozen than experimental example, and the cell of more than 90% all has normal chromosome number.
4, fluorescence immunoassay cell dyeing analyzes the expression of recovery CK5 and 8
Keratin 5 and CK8 are the marks of galactophore epithelial cell, and wherein keratin 5 is expressed in the basal layer cell of all stratified epithelium, and CK8 is at the chamber epithelial secretion cells of mammary gland.The method of application SABC detects the expression of recovery CK5 and CK8, is the important indicator whether detection cell meets galactophore epithelial cell feature.Concrete steps are as follows: cell to be detected at room temperature fixes 30min with 4% paraformaldehyde, then use the PBS of 0.2%TritonX-100 penetrating process cell 5min, and PBS cleans 3 times, each 5min.With the PBS Seal treatment 30min containing 10% lowlenthal serum.Then oscillation incubation 1h, PBS clean 3 times at 37 DEG C to use the CK5 of mouse-anti people and CK8 monoclone antibody (1:50 dilution), each 5min.Add the goat anti-mouse IgG antibody (1:50) containing Cy3 mark, 37 DEG C of lucifuge oscillation incubation 30min, PBS cleans 3 times, each 5min.Add the PBS containing iodate third ingot 10 μ g/mL again, under room temperature, contaminate core 10min.Use fluorescence microscope result, do negative control with sheep fibroblast simultaneously.Result shows, as shown in Figure 5, and the basalis like cell that keratin 5 dyeing is less, the differentiation chamber like cell that CK8 dyeing is larger, result proves have a large amount of keratin 5s and CK8 in recovery cell cytosol, and the sheep fibroblast as negative control is then negative, as shown in Figure 6.This result proves: the cell after recovery all can ensure that galactophore epithelial cell biological property is constant.
5, real-time quantitative PCR detects the expression of beta-casein gene
The cells containing sequences expression-secretion milk protein of culture in vitro, this is significant to studying lactation mechanism and detecting mammary gland protein carrier.Therefore, the expression carrying out beta-casein gene to the galactophore epithelial cell of recovery detects the important indicator that can keep its biological function when being and detecting recovery cell.
Trizol (Invitrogen) one-step method is utilized to extract total serum IgE from the Goats Milk glandular epithelium of recovery, then its reverse transcription is become cDNA, utilize milch goat beta-casein gene as shown in table 2 and β-actin gene primer to carry out pcr amplification and whether express beta-casein mRNA to detect cell.Reaction system is as follows:
MJ MiniOpticon System PCR instrument is carried out, and reaction condition is specially
Get PCR primer and carry out 1% agarose gel electrophoresis detection, after Preliminary Identification is correct, send PCR primer to carry out sequencing analysis.By reverse transcription PCR technology, can directly detect the expression of beta-casein gene mRNA in cell, electrophoresis result is presented at 300bp place and occurs object band, confirms that amplified production is beta-casein gene through sequencing analysis.Utilize relative quantification pcr analysis beta-casein gene at the expression of the galactophore epithelial cell of recovery, as shown in Table 3, the expression of the beta-casein gene in contrast experiment's example is extremely low, and the galactophore epithelial cell beta-casein gene expression in embodiment 1, embodiment 2 and embodiment 3 is 10.3 times, 11.6 times and 13.4 times of contrast experiment's example respectively.As can be seen here, insulin-transferrins-selenium sodium not only effectively can retain the biological property of galactophore epithelial cell, more can promote that galactophore epithelial cell keeps the secreting function of milk protein.
Table 2 Xinong Saanen goat beta-casein and beta-actin primer sequence
The relative quantitative assay of table 3 beta-casein gene in Goat Mammary Epithelial Cells
Claims (5)
1. preserve the epithelioglandular cryopreserving liquid of Goats Milk, it is characterized in that, mixed 100:1-2 by volume by basic cryopreserving liquid and insulin-transferrins-selenium sodium;
Described basic cryopreserving liquid by percent by volume be the dimethyl sulfoxide (DMSO) of 10%, the DMEM/F12 medium of 30-50% and 40 – 60% hyclones mix, above-mentioned three components percent by volume summation is 100%.
2. the cryopreservation methods preserving the epithelioglandular cryopreserving liquid of Goats Milk as claimed in claim 1, is characterized in that, comprise the following steps:
(1), the Goats Milk glandular epithelium in vegetative period of taking the logarithm, add mass concentration be 0.25% trypsase and mass concentration be 0.02% EDTA mixed solution, be placed in 37 DEG C, 5%C0
2constant incubator in digest, until cytoplasm retraction, space between cells increase, cell shorten circle into time, with containing percent by volume 10% hyclone termination medium stop digestion, centrifugal, supernatant discarded, obtains Goats Milk glandular epithelium;
(2), gained Goats Milk glandular epithelium adds described cryopreserving liquid, and piping and druming evenly, forms cell suspension, cell suspension is placed in cryopreservation tube;
(3), by above-mentioned cryopreservation tube be closed in foam freezing storing box, foam freezing storing box is placed 15h under-80 DEG C of cryogenic conditions;
(4), preserve in foam freezing storing box immersion liquid nitrogen.
3. utilize the cryopreservation methods of the epithelioglandular cryopreserving liquid of preservation Goats Milk described in claim 1 as claimed in claim 2, it is characterized in that, in described step (1), centrifugal rotational speed is 1500rpm, centrifugation time 10min.
4. utilize the cryopreservation methods of the epithelioglandular cryopreserving liquid of preservation Goats Milk described in claim 1 as claimed in claim 2, it is characterized in that, in described step (2), the cell concentration of cell suspension is 0.5-1 × 10
7individual/mL.
5. utilize the cryopreservation methods of the epithelioglandular cryopreserving liquid of preservation Goats Milk described in claim 1 as claimed in claim 2, it is characterized in that, the upper surface of described foam freezing storing box is porose, the degree of depth in hole is greater than the height of described cryopreservation tube, described cryopreservation tube inserts in the hole, and aperture foam block is closed.
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| CN104946579A (en) * | 2015-07-03 | 2015-09-30 | 浙江大学 | Culture method of Jinhua pig mammary gland epithelial cell line |
| 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 |
| CN108293981A (en) * | 2018-03-07 | 2018-07-20 | 贵州大学 | A kind of attached tumor cells cryopreservation methods and improvement freeze formula of liquid |
| CN113331176A (en) * | 2021-06-01 | 2021-09-03 | 北京戴域生物技术有限公司 | Mesenchymal stem cell cryopreservation liquid |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104946579A (en) * | 2015-07-03 | 2015-09-30 | 浙江大学 | Culture method of Jinhua pig mammary gland epithelial cell line |
| 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 |
| CN108293981A (en) * | 2018-03-07 | 2018-07-20 | 贵州大学 | A kind of attached tumor cells cryopreservation methods and improvement freeze formula of liquid |
| CN113331176A (en) * | 2021-06-01 | 2021-09-03 | 北京戴域生物技术有限公司 | Mesenchymal stem cell cryopreservation liquid |
| CN113331176B (en) * | 2021-06-01 | 2022-04-29 | 零下十八度(北京)生物科技有限公司 | Umbilical cord mesenchymal stem cell cryopreservation liquid |
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