CN105754933A - Cell in-vitro culture method for primary culture of bovine mammary epithelial cells - Google Patents
Cell in-vitro culture method for primary culture of bovine mammary epithelial cells Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
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Abstract
本发明公开了用于奶牛乳腺上皮细胞原代培养的细胞体外培养方法，是将奶牛乳腺组织预处理后采用培养瓶侧置法静置贴壁培养30min，再配合组织块回收法进行分离培养，最后采用差时胰酶消化法配合瓶内消化法纯化获得纯化的奶牛乳腺上皮细胞。本发明的有益效果为：本发明提供的用于奶牛乳腺上皮细胞原代培养的细胞体外培养方法，成功获得纯化的奶牛乳腺上皮细胞，同时，使细胞爬出时间缩短，细胞增殖速度提高，细胞生长状态良好，大大降低了细胞污染的可能性，避免了采用泌乳期乳腺组织初乳不易清洗且容易污染的问题。The invention discloses an in vitro cell culture method for primary culture of dairy cow mammary gland epithelial cells. After pretreatment, the milk cow mammary gland tissue is placed on the side of a culture bottle for standing and adhering to the wall for 30 minutes, and then combined with a tissue block recovery method for separation and culture. Finally, the purified dairy cow mammary gland epithelial cells were purified by time-difference trypsinization and in-flask digestion. The beneficial effects of the present invention are: the cell in vitro culture method for the primary culture of dairy cow mammary gland epithelial cells provided by the present invention successfully obtains purified dairy cow mammary gland epithelial cells, and at the same time shortens the time for cells to climb out, increases the speed of cell proliferation, and The growth state is good, the possibility of cell contamination is greatly reduced, and the problem of using colostrum of lactating mammary gland tissue that is difficult to clean and easy to contaminate is avoided.
Description
Technical field
The present invention relates to cell engineering field, be particularly used for the cell injuring model method of cow mammary gland epithelial cells original cuiture.
Background technology
Galactophore epithelial cell is the cell uniquely in mammary gland with secretory function, it it is the basis realizing lactogenic, also it is the important component part constituting mammary gland innate immunity barrier simultaneously, no matter primary cell is morphosis or physio-biochemical characteristics aspect, can the good truth in antimer, it is suitable for the research of the aspects such as physiology, pathology, pharmacology, galactophore epithelial cell has been used for research lactogenic mechanism, mastitis pathogenesis and drug screening etc., therefore this cell becomes the important tool of research lactogenic mechanism, mastitis pathogenesis and drug screening.Extensive use along with tissue culture technique, cow mammary gland epithelial cells Vitro Culture Techniques have also been obtained and develops rapidly and apply, this technology is it can be avoided that in vivo test cycle length, cost are high, complicated condition and there are the problems such as individual variation, so obtaining the target that the cow mammary gland epithelial cells that cell state is good, multiplication capacity is strong is numerous research worker when cultivating in vitro.Current cow mammary gland epithelial cells original cuiture mainly has four kinds of methods, respectively tissue mass cell culture, mechanical crushing method, enzyme digestion and milk partition method.But all there are some defects in these methods.Tissue mass cell culture advantage is that cost growth and proliferation of cell ability low, simple to operate, that obtain is stronger；But a disadvantage is that length consuming time, subsequent purification work difficulty is big.The advantage of mechanical crushing method be simple to operate, cost is low, cell yield is higher and growth rapidly；Shortcoming is cell to have certain mechanical damage and follow-up needs purification.The advantage of enzyme digestion is that cell proliferation is very fast and purity is higher；Shortcoming is that cell can be caused certain damage by enzyme, so strictly to control concentration and the digestion time of enzyme in operation.The advantage of milk extraction method be in that the cell purity obtained higher, active good, required time is short, but the method still exists some problems, quality and quantitative requirement if desired for the paying attention sampling time, to milk cell quantity higher, that obtain is less, have partial immunity cell contamination etc..So for the cow mammary gland epithelial cells being successfully obtained purification in vitro, those skilled in the art not only need to improve in method, and it is also required to update in the selection of tissue and in the details of operation.
Summary of the invention
The purpose of the present invention is aiming at above-mentioned defect of the prior art, provide a kind of cell injuring model method for cow mammary gland epithelial cells original cuiture, the mammary gland tissue position not only determining cow mammary gland epithelial cells original cuiture selects, and original extracorporeal culturing method has been improved.
To achieve these goals, technical scheme provided by the invention is: for the cell injuring model method of cow mammary gland epithelial cells original cuiture, it is culture bottle side method will be adopted after cow mammary gland Antigen repairing to stand adhere-wall culture 30min, coordinate piece of tissue absorption method to be easily separated cultivation again, finally adopt the cow mammary gland epithelial cells of trypsin digestion cooperation bottle digested method purification acquisition purification during difference.
Further, the above-mentioned cell injuring model method for cow mammary gland epithelial cells original cuiture, described cow mammary gland is organized as and reaches sexual maturity, the middle part of mammary gland tissue of milch cow without lactogenic history.
Further, the above-mentioned cell injuring model method for cow mammary gland epithelial cells original cuiture, the preprocess method of described cow mammary gland tissue is: takes tissue in the middle part of cow mammary gland and is placed in the ethanol of 75% and soaks 3min, embathe 3～4 times with physiological saline solution and remove ethanol；The tissue of the outside whiting of mammary gland tissue, fatty tissue and connective tissue are rejected by sterile working, namely the parenchymal tissue of galactophore epithelial cell is obtained, then put in the beaker equipped with appropriate D-Hank ' s liquid after rinsing 3～4 times with D-Hank ' s liquid, parenchymal tissue is cut into 1mm3Fritter；With D-Hank ' s liquid, the fritter parenchymal tissue shredded is cleaned up, discard the tissue debris swimming in top, with 5mL rifle head aspirates tissue block pastel；The pasty state tissue that will draw, at the bottom of being laid on bottle, in the same size, it is uniformly distributed, adopts culture bottle side method standing adhere-wall culture 30min.
Further, the above-mentioned cell injuring model method for cow mammary gland epithelial cells original cuiture, the operating process of described piece of tissue absorption method is: take out the culture bottle after above-mentioned side method stands adhere-wall culture 30min, surplus liquid at the bottom of bottle is absorbed with suction pipe, liquid-transfering gun is used to be slowly added to complete culture solution at bottleneck, it is annihilated piece of tissue, puts into 37 DEG C, 5%CO in CO2 gas incubator2Cultivating, culture fluid changes fresh medium when turning yellow in time；Until at the bottom of cell will be paved with bottle time, remove piece of tissue, and the piece of tissue removed is re-seeded in new culture bottle, cultivate in incubator and change fresh medium in time.
Further, the above-mentioned cell injuring model method for cow mammary gland epithelial cells original cuiture, the preparation method of described complete culture solution is: in DMEM/F12 basic culture solution, the ratio of 5:1 adds hyclone FBS by volume, it is subsequently adding antibacterial medicines, wherein the content of penicillin is 500U/mL, the content of streptomycin is 500U/mL, and the content of amphotericin B is 5 μ g/mL.
Further, the above-mentioned cell injuring model method for cow mammary gland epithelial cells original cuiture, described poor time trypsin digestion coordinate the digested method purification cow mammary gland epithelial cells of bottle specific operation process be: difference time trypsin digestion, cell obtained above is discarded culture fluid, after D-Hank ' s cleans, add 1mL trypsinization liquid, digestion 30～60s, rock culture bottle gently, basis of microscopic observation, treat that fibroblast shrinks to become round to float, galactophore epithelial cell has when shrinking but do not float, add 2mL complete culture solution immediately and terminate digestion, complete culture solution is discarded after rocking gently, add 4mLD-Hank ' s liquid, the fibroblast that eccysis is remaining gently, add complete culture solution in cell culture incubator 37 DEG C, 5%CO2Cultivate；The digested method of bottle, it is in the process of trypsin digestion purification cow mammary gland epithelial cells, carry out the digested tiling of bottle: add 4mLD-Hank ' s liquid, after cleaning when difference, add 1mL Digestive system, after digestion 2～3.5min, discard 600 μ L Digestive systems, pat from bottom at the bottom of bottle and make cell completely fall off, add 4mL complete culture solution and terminate digestion, repeatedly blow and beat cell, make cell mass be dispersed into individual cells, be beneficial to cultivation.
The invention have the benefit that a kind of cell injuring model method for cow mammary gland epithelial cells original cuiture provided by the invention, it is successfully obtained the cow mammary gland epithelial cells of purification, simultaneously, cell is made to climb out of time shortening, cell proliferation rate improves, cell growth state is good, greatly reduces the probability of cell contamination, it is to avoid adopt mammary gland tissue colostrum not easy cleaning and the problem easily polluted lactation period.
Accompanying drawing explanation
Fig. 1 is that culture bottle puts method schematic diagram.
Wherein, A is side method, and B is anastrophe.
Fig. 2 is after adopting tissue block method, different parts tissue cell growth situation map.
Wherein, A1 is that the 4th day cell growth status is organized on top, and A2 is that the 5th day cell growth status is organized on top, and A3 is that the 6th day cell growth status is organized on top；B1 is that the 4th day cell growth status is organized at middle part, and B2 is that the 5th day cell growth status is organized at middle part, and B3 is that the 6th day cell growth status is organized on top；C1 is that the 4th day cell growth status is organized in bottom, and C2 is that the 5th day cell growth status is organized on top, and C3 is that the 6th day cell growth status is organized on top.
Fig. 3 is comparison diagram before and after the purification of cow mammary gland epithelial cells.
Wherein, A is the cell (1 is lipochondrion, and 2 is the bMEC before purification, and 3 is the fibrocyte before purification) before purification；B is the cow mammary gland epithelial cells after purification.
Fig. 4 is the qualification result figure of the Keratin 18 of cow mammary gland epithelial cells.
Wherein A is fluorescently-labeled cytokeratin-18；B is nuclear targeting；C be A figure and B figure merge figure.
Detailed description of the invention
Embodiment 1:
1, laboratory animal:
Experimental animal is that 24 monthly age health are bred as holstein cow, without lactogenic history.
2, overall mammary gland tissue is gathered:
After test milch cow is butchered, breast surface clear water fully rinses, 75% alcohol disinfecting, and autoclaving dissecting knife cuts open from whole breast, is placed in the sampling box that 75% ethanol postincubation is crossed, takes back laboratory in 1h.
3, cell cultivation with mammary gland tissue process "
Again with 75% alcohol disinfecting tissue surface, avoid fatty tissue, connective tissue and blood vessel, the mammary gland essence of clip organization internal, about 2.5cm3, tissue clip position is as follows respectively:
Top tissue: tissue is in yellow, and matter is close, obvious with peripheral adipose tissue boundary, duct free.
Middle part tissue: tissue, in yellow, compares matter close, has visible tiny conduit.
Bottom tissue: tissue is in yellow, more open, has thicker tracheal tissue, and clip presses close to the mammary gland parenchymal tissue of conduit.
It is transferred to iuntercellular after the alcohol-pickled 3min of tissue 75% gathered.
4, galactophore epithelial cell tissue block method separation and Culture:
In each culture bottle, add Collagen type-I in advance, cover at the bottom of whole bottle, irradiate 4h under uviol lamp, outwell collagen before inoculation, clean 3 times；The piece of tissue processed is taken out by aseptic operating platform from the ethanol of 75%, embathes 3～4 times to remove ethanol with physiological saline solution；The tissue of the outside whiting of above-mentioned mammary gland tissue, fatty tissue and connective tissue are rejected by sterile working, namely the parenchymal tissue of galactophore epithelial cell is obtained, then put into the small beaker equipped with appropriate D-Hank ' s liquid after rinsing 3～4 times with D-Hank ' s liquid, parenchymal tissue is cut into about 1mm3Fritter；With D-Hank ' s liquid, the fritter parenchymal tissue shredded is cleaned up, discard the tissue debris swimming in top, with 5mL rifle head aspirates tissue block pastel, be advisable not blocking rifle head；The pasty state tissue that will draw, at the bottom of being laid on bottle, in the same size, it is uniformly distributed, as it is shown in figure 1, be respectively adopted culture bottle side method and 2 kinds of methods of anastrophe, time of repose 30min and 1h2 the time of employing；Taking out culture bottle, absorb surplus liquid at the bottom of bottle with suction pipe, use liquid-transfering gun to be slowly added into complete culture solution at bottleneck, piece of tissue of being just annihilated is preferred, and puts in incubator and cultivates, and culture fluid to change fresh culture fluid when turning yellow in time；Until at the bottom of cell is almost paved with bottle time, removes piece of tissue, and the piece of tissue removed is re-seeded in new culture bottle, cultivate in incubator and change liquid in time.Top tissue, middle part tissue, bottom tissue carry out cell cultivation according to above method respectively, and carry out labelling, follow the tracks of and observe under microscope.As a result, in the identical time, the adherent effect of culture bottle side method tissue is better than anastrophe, stands 30min and adherent best results.The uterus tissue pieces that side processes climbs out of to the 4th～5 day visible cell, and the uterus tissue pieces that inversion processes climbs out of to the 6th day visible cell；Climb out of in fibroblast after cobblestone sample galactophore epithelial cell, have obvious boundary between the two；Portion of tissue can directly climb out of cobblestone sample epithelial cell, climbs out of without fibroblast.Within 2nd day, namely there is cell to climb out of after the tissue inoculation reclaimed, and cobblestone like cell ratio is higher.Tissue block method's observed result shows, top tissue not easily climbs out of cell and cell proliferation rate relatively slow (in Fig. 2 A1-A3)；Middle part tissue easily directly climbs out of, and vitro growth rates very fast (in Fig. 2 B1-B3)；Bottom tissue grows cell and high cell growth speed at first, but major part is fibroblast, and the later stage grows galactophore epithelial cell, has comparatively significantly demarcation line (in Fig. 2 C1-C3) with fibroblast contact site.
When 5, adopting poor, trypsin digestion coordinates the digested method purification of bottle to obtain cow mammary gland epithelial cells:
Trypsin digestion during difference: cell discards culture fluid, after D-Hank ' s cleans, adds 1mL trypsinization liquid, about 30～60s, rocks culture bottle, basis of microscopic observation gently, fibroblast shrinks to become round and floats, and galactophore epithelial cell slightly shrinks, and does not float, add 2mL complete culture solution immediately and terminate digestion, and discard complete culture solution, add 4mLD-Hank ' s liquid, the fibroblast that eccysis is remaining gently, add complete culture solution to cultivate in cell culture incubator (37,5%CO2).Every day examines under a microscope cell, as little in cell density, just looks in cell still residual fraction fibroblast, then repeats trypsin digestion during difference, and purification can obtain purer cow mammary gland epithelial cells 2～3 times.
The digested method of bottle: when difference in the process of trypsin digestion purification, if basis of microscopic observation cell, if it find that galactophore epithelial cell is sheet assembles growth, it is unfavorable for cell proliferation, the digested tiling of bottle can be carried out: add 4mLD-Hank ' s liquid, after cleaning, add 1mL Digestive system, after digestion about 2～3.5min, discard about 600 μ L Digestive systems, then pat from bottom flat, make cell completely fall off, add 4mL complete culture solution, terminate digestion, repeatedly blow and beat cell, make cell mass be dispersed into individual cells, cultivate.
When adopting poor, trypsin digestion coordinates the digested method purification of bottle, final cow mammary gland epithelial cells (Fig. 3) that obtain purification, that growth conditions is good.
6, cow mammary gland epithelial cells is identified:
The process of coverslip: take after coverslip cleans through acid treatment 48h, fully rinse well, be then soaked in dehydrated alcohol.Bottom 6 porocyte culture plates, drip culture fluid, clamping cover slide, after ethanol volatilizees, put into 6 porocyte culture plates gently；Cell is adjusted and is inoculated in 6 orifice plates to debita spissitudo, wait cell monolayer to remove culture fluid when being fused to 80%, then wash cell 2 times with D-Hank ' s solution, each 5min；With the fixing cell of 4% paraformaldehyde of pre-cooling, 10min at 4 DEG C, remove paraformaldehyde, clean cell with TBSTx solution, be placed on shaking table and be shaken gently for, each 5min, repeat 3 times, edge moisture cleaned by last filter paper；With the TBSTx confining liquid submergence cell climbing sheet containing 5%BSA, close 1h for 37 DEG C；Removing confining liquid, the primary antibodie of the Keratin 18 TBSTx containing 5%BSA is diluted 50 times, dropping, on cell climbing sheet, hatches 1h for 37 DEG C；Carefully absorb primary antibodie, with TBSTx, cell is washed 3 times, shaking table slowly shakes, each 5min；What cell was dipped in the FITC labelling after TBSTx containing 5%BSA dilutes 100 times two resists, and hatches 60min for 37 DEG C；Careful absorption two resists, and cleans cell 3 times with TBSTx, shaking table slowly shakes, each 5min；1 μ g/mlPI dyeing liquor is added drop-wise on cell climbing sheet, incubated at room temperature 15min；Suck PI dyeing liquor, clean cell 3 times with TBSTx, shaking table slowly shakes, each 5min；Taking clean glass slide, central authorities dropping 1-2 drips fluorescence quenching, takes out cell climbing sheet, and the side that length has cell is downward, carries out mounting at fluorescence microscopy Microscopic observation.As shown in Figure 4, it is shown that the specific expressed Keratin 18 of galactophore epithelial cell, and its nucleus position essentially coincides with nuclear targeting position, have successfully been obtained the cow mammary gland epithelial cells of purification.
Last it is noted that the foregoing is only the preferred embodiments of the present invention, it is not limited to the present invention, although the present invention being described in detail with reference to previous embodiment, for a person skilled in the art, technical scheme described in foregoing embodiments still can be modified by it, or wherein portion of techniques feature carries out equivalent replacement.All within the spirit and principles in the present invention, any amendment of making, equivalent replacement, improvement etc., should be included within protection scope of the present invention.
Claims (6)
1. for the cell injuring model method of cow mammary gland epithelial cells original cuiture, it is characterized in that, it is culture bottle side method will be adopted after cow mammary gland Antigen repairing to stand adhere-wall culture 30min, coordinate piece of tissue absorption method to be easily separated cultivation again, finally adopt the cow mammary gland epithelial cells of trypsin digestion cooperation bottle digested method purification acquisition purification during difference.
2. the cell injuring model method for cow mammary gland epithelial cells original cuiture according to claim 1, it is characterised in that described cow mammary gland is organized as and reaches sexual maturity, the middle part of mammary gland tissue of milch cow without lactogenic history.
3. the cell injuring model method for cow mammary gland epithelial cells original cuiture according to claim 1, it is characterized in that, the preprocess method of described cow mammary gland tissue is: takes tissue in the middle part of cow mammary gland and is placed in the ethanol of 75% and soaks 3min, embathe 3～4 times with physiological saline solution and remove ethanol；The tissue of the outside whiting of mammary gland tissue, fatty tissue and connective tissue are rejected by sterile working, namely the parenchymal tissue of galactophore epithelial cell is obtained, then put in the beaker equipped with appropriate D-Hank ' s liquid after rinsing 3～4 times with D-Hank ' s liquid, parenchymal tissue is cut into 1mm3Fritter；With D-Hank ' s liquid, the fritter parenchymal tissue shredded is cleaned up, discard the tissue debris swimming in top, with 5mL rifle head aspirates tissue block pastel；The pasty state tissue that will draw, at the bottom of being laid on bottle, in the same size, it is uniformly distributed, adopts culture bottle side method standing adhere-wall culture 30min.
4. the cell injuring model method for cow mammary gland epithelial cells original cuiture according to claim 1, it is characterized in that, the operating process of described piece of tissue absorption method is: take out the culture bottle after the side method described in claim 3 stands adhere-wall culture 30min, surplus liquid at the bottom of bottle is absorbed with suction pipe, liquid-transfering gun is used to be slowly added to complete culture solution at bottleneck, it is annihilated piece of tissue, puts into 37 DEG C, 5%CO in CO2 gas incubator2Cultivating, culture fluid changes fresh medium when turning yellow in time；Until at the bottom of cell will be paved with bottle time, remove piece of tissue, and the piece of tissue removed is re-seeded in new culture bottle, cultivate in incubator and change fresh medium in time.
5. the cell injuring model method for cow mammary gland epithelial cells original cuiture according to claim 4, it is characterized in that, the preparation method of described complete culture solution is: in DMEM/F12 basic culture solution, the ratio of 5:1 adds hyclone FBS by volume, it is subsequently adding antibacterial medicines, wherein the content of penicillin is 500U/mL, the content of streptomycin is 500U/mL, and the content of amphotericin B is 5 μ g/mL.
null6. the cell injuring model method for cow mammary gland epithelial cells original cuiture according to claim 1，It is characterized in that，Described poor time trypsin digestion coordinate the digested method purification cow mammary gland epithelial cells of bottle specific operation process be: difference time trypsin digestion，Cell claim 4 obtained discards culture fluid，After D-Hank ' s cleans，Add 1mL trypsinization liquid，Digestion 30～60s，Rock culture bottle gently，Basis of microscopic observation，Treat that fibroblast shrinks to become round to float，Galactophore epithelial cell has when shrinking but do not float，Add 2mL complete culture solution immediately and terminate digestion，Complete culture solution is discarded after rocking gently，Add 4mLD-Hank ' s liquid，The fibroblast that eccysis is remaining gently，Add complete culture solution in cell culture incubator 37 DEG C、5%CO2Cultivate；The digested method of bottle, it is in the process of trypsin digestion purification cow mammary gland epithelial cells, carry out the digested tiling of bottle: add 4mLD-Hank ' s liquid, after cleaning when difference, add 1mL Digestive system, after digestion 2～3.5min, discard 600 μ L Digestive systems, pat from bottom at the bottom of bottle and make cell completely fall off, add 4mL complete culture solution and terminate digestion, repeatedly blow and beat cell, make cell mass be dispersed into individual cells, be beneficial to cultivation.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| CN106811440A (en) * | 2017-02-09 | 2017-06-09 | 广西壮族自治区水牛研究所 | The method of buffalo galactophore epithelial cell is separately cultured in a kind of milk from buffalo |
| CN110592019A (en) * | 2018-06-13 | 2019-12-20 | 北京吉尚立德生物科技有限公司 | Dissociation liquid for colorectal cancer solid tumor tissue sample |
| CN111808816A (en) * | 2019-04-11 | 2020-10-23 | 北京基石生命科技有限公司 | A medium for culturing gastric cancer solid tumor primary cells |
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| CN101591636A (en) * | 2008-05-26 | 2009-12-02 | 新疆维吾尔自治区畜牧科学院中国-澳大利亚绵羊育种研究中心 | The extracorporeal culturing method of separating mammary epithelial cells from bovine coloctrum |
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| CN101591636A (en) * | 2008-05-26 | 2009-12-02 | 新疆维吾尔自治区畜牧科学院中国-澳大利亚绵羊育种研究中心 | The extracorporeal culturing method of separating mammary epithelial cells from bovine coloctrum |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN106811440A (en) * | 2017-02-09 | 2017-06-09 | 广西壮族自治区水牛研究所 | The method of buffalo galactophore epithelial cell is separately cultured in a kind of milk from buffalo |
| CN106811440B (en) * | 2017-02-09 | 2020-09-15 | 广西壮族自治区水牛研究所 | Method for separating and culturing buffalo mammary epithelial cells from buffalo milk |
| CN110592019A (en) * | 2018-06-13 | 2019-12-20 | 北京吉尚立德生物科技有限公司 | Dissociation liquid for colorectal cancer solid tumor tissue sample |
| CN111808816A (en) * | 2019-04-11 | 2020-10-23 | 北京基石生命科技有限公司 | A medium for culturing gastric cancer solid tumor primary cells |
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