CN102505005B - Culture medium for keeping primary airway epithelial cells in physiological state in vivo - Google Patents
Culture medium for keeping primary airway epithelial cells in physiological state in vivo Download PDFInfo
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- CN102505005B CN102505005B CN 201110440894 CN201110440894A CN102505005B CN 102505005 B CN102505005 B CN 102505005B CN 201110440894 CN201110440894 CN 201110440894 CN 201110440894 A CN201110440894 A CN 201110440894A CN 102505005 B CN102505005 B CN 102505005B
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Abstract
The invention relates to preservation of human local living parts and in particular relates to a culture medium for keeping primary airway epithelial cells in the physiological state in vivo during in vitro culture. The invention is characterized in that the culture medium contains the following components (per liter), 12 g of DMEM/F12 medium, 30 mg of penicillin G sodium salt, 50 mg of streptomycin, 10 mg of insulin, 5 mg of transferring, 25 Mug of epidermal growth factors, 100 Mug of cholera toxin, 108.741 Mug of hydrocortisone, 1 g of bovine pituitary extract, 3 g of bovine serum albumin, 1.5*10<-2> Mug of retinoic acid, 50 mg of gentamycin, 0.5 mg of amphotericin B, 50 mL of fetal calf serum and the balance of water. The primary airway epithelial cells cultured by the culture medium have the ciliary beating function and can maintain the physiological state in vivo.
Description
Technical field
The present invention relates to the preservation of the local live body part of people, and in particular to the culture medium of primary airway epithelial cell.
Background technology
Airway epithelial cell constitutes first of natural cover for defense from nasal cavity to bronchioli terminales, is the main starting position that inflammatory reaction is produced after pathogen invades body through respiratory tract.Use A549 mostly both at home and abroad, 16HBE, the cell line such as BEAS-2B replicates the cell model of breathing problem, to microorganisms infection and inflammatory reaction etc..But A549, this kind of cell line such as 16HBE derives from tumour cell in itself, or obtaining immortalizing after being merged with tumour cell by eupnea tract epithelial cell such as BEAS-2B turns into cell line, their physiological characteristic generates certain limitation with being differed greatly in body cell to the cytology research of breathing problem.Therefore, original cuiture airway epithelial cell, and make it physiologically close in body state, with important value.
Airway epithelial cell is attached to basement membrane growth, collectively forms the pseudostratified columnar ciliated epithelium of respiratory tract, and the cilium on its surface has played important function with having the rhythm and pace of moving things to pharyngeal motor during body excludes respiratory tract irritation foreign matter.Basement membrane is the film between epithelial cell basal surface and the connective tissue in deep, is made up of following two parts：Turn into substrate close to the part of epithelium, be mainly made up of laminins, collagen and heparin sulfate proteoglycan etc.；The part connected with connective tissue is web plate.Respiratory tract is directly communicated with the external world, airway epithelial cell is had more chance contact pathogenic microorganism, it, which is grown in microenvironment, has powerful anti-infection bio factor.Due to easily being damaged by environmental stimuli, to maintain to be rich in some stimulating factors in stable state, local environment, they, which act on airway epithelial cell, makes it possess vigorous self-renewal capacity.Due to being limited by this special growth conditions, and existing conventional medium has not been able to build the microenvironment of suitable airway epithelial cell growth, and this kind of cell primary culture is more difficult.
The content of the invention
It is to provide a kind of culture medium of primary airway epithelial cell that the technical problems to be solved by the invention, which are, and the primary airway epithelial cell that the culture medium is cultivated has fibre swing function, keeps it in body physiological status.
The technical scheme of problem is in present invention solution：
A kind of culture medium for the primary airway epithelial cell for being maintained at body physiological status, it is characterised in that every liter of culture medium is composed of the following components：DMEM/F12 culture medium 12g, penicillin G sodium salt 30mg, streptomysin 50mg, insulin 10mg, transferrins 5mg, the μ g of EGF 25, the μ g of cholera toxin 100, the μ g of hydrocortisone 108.741, ox pituitary extract 1g, bovine serum albumin(BSA) 3g, vitamin A acid 1.5 × 10-2μ g, gentamicin 50mg, amphotericin B 0.5mg, hyclone 50ml, remaining is water.
DMEM/F12 culture mediums in such scheme are the product that invitrogen companies of the U.S. produce, and its product designation is 12500-096, and the constituent of product is shown in product description.
The preparation method of culture medium of the present invention is as described below：
Penicillin, streptomysin, insulin, transferrins, EGF, cholera toxin, hydrocortisone, ox pituitary extract, bovine serum albumin(BSA), vitamin A acid, gentamicin, amphotericin B and hyclone are added in DMEM/F12 culture mediums by proportioning, add water to 1 liter of fully mixing, 0.22 μm of filtering with microporous membrane of laminar flow cell culture chamber, collects filtrate.
In culture medium of the present invention, nutrient solution constitutes the basic microenvironment of cell development based on DMEM/F12；Insulin can be by promoting cellular uptake glucose sugar and amino acid, so as to promote cell multiple fission；Transferrins can combine iron ion, reduce its toxicity and utilized by cell, while fibroblastic quantity in incubation can be reduced；EGF has strong rush splitting action to airway epithelial cell；Ox pituitary extract contains the composition of a variety of promotion cell growths, bovine serum albumin(BSA) then can provide carrier for a variety of growth factors including EGF, the presence of these growth stimulants, more effectively have stimulated the growth of cell, improve the culture effect of cell；Cholera toxin and vitamin A acid have the function of promoting cell proliferation and differentiation and enhancing injury repair respectively；Hydrocortisone can help to growing for airway epithelial cell；Penicillin, streptomysin, gentamicin and amphotericin B together constitute powerful defensive barrier, have potent inhibitory action to fungi, Gram-positive and negative bacteria, reduce pollution risk.The synergy of above-mentioned each component, making the primary airway epithelial cell of culture has fibre swing function, remain primary airway epithelial cell in body physiological status.
Foreign countries think that serum can suppress the growth of airway epithelial cell, therefore generally use free serum culture at present.And not only the result of study of the present inventor on the contrary, help somewhat to cell attachment on the contrary containing hyclone in nutrient solution to the no inhibitory action of the growth of airway epithelial cell.
In addition, the primary airway epithelial cell growth vigor cultivated using culture medium of the present invention is good.At present, commercially available primary airway epithelial cell is expensive, and most rhythmic movements for all having lost fibre swing, illustrates that its physiological status differs larger with body level.Instant invention overcomes disadvantages mentioned above, and prepare simple, repeatability is strong, and operation is simple, easy to utilize.The present invention provides favourable material conditions for the scientific research, teaching, medical application about airway epithelial cell, with extensive promotional value.
Brief description of the drawings
The hologram for the primary airway epithelial cell that Fig. 1 is cultivated by 30 following embodiments being continuously shot under light microscope.
Embodiment
First, culture medium is prepared
For the ease of storage and experimental implementation, bought chemical reagent is first configured to normal storage concentration by this experiment, preserved for a long time under suitable temperature conditionss, therefore the consumption of each component calculates consumption in addition to hyclone by made normal storage concentration in culture medium described in this example.Specific preparation process is as described below：
Storage concentration shown according to the form below 1 and it is added to penicillin, streptomysin, insulin, transferrins, EGF, cholera toxin, hydrocortisone, ox pituitary extract, bovine serum albumin(BSA), vitamin A acid, gentamicin, amphotericin B and hyclone is measured in DMEM/F12 culture mediums, 100ml is added water to fully to mix, 0.22 μm of filtering with microporous membrane of laminar flow cell culture chamber, collects filtrate and produces 100ml culture mediums of the present invention.
Table 1
2nd, primary airway epithelial cell is cultivated
1st, culture dish collagen is coated with
Using preceding 24h, by 3.0mg/ml type i collagens: 60g/L DMEM/F12: 0.5mol/LNaOH=4ml: 1ml: 0.15ml proportions collagen mixed solution (collagen solution meta-acid, adds certain density NaOH, and regulation pH value is 7.2 or so).The orifice plate of cell culture 24 is disposed vertically 30min on ice, and 250 μ l collagen mixed solutions are added dropwise per hole, and 37 DEG C are incubated 10min and solidify it；Then continue additional 150 μ l collagen mixed solutions, 37 DEG C are incubated at least 1h and make bottom glue surfacing, the culture medium obtained by 0.5ml steps one is finally added again, is completed, 4 DEG C save backup.
2. mouse breathing tract epithelial cell is separately cultured
The separation of 2.1 mouse tracheaes
BALB/c mouse is taken through CO2Asphyxia is put to death, and is fixed on operating table, tilts head, in its thorax abdomen alcohol disinfecting, notes protection oral cavity and nasal meatus, is stimulated from alcohol.The downward hunter's line of aseptic condition cuts off skin, and exposure abdominal aorta cuts bloodletting to flowing to end, its object is to avoid being mixed into excessive red blood cell during separation airway epithelial cell；Blunt separation muscle, exposure tracheae, and it is fully dissociated；Bifilar No. 5 surgical threads are worn, a untwisting is made a call to tracheae upper and lower side is each, ties edge otch in the upper end, wear hollow tubing conductor, deeply after about 5mm, conduit is simultaneously fixed on tracheal strips by urgent original untwisting；Cut between the knot and tracheae crotch of lower end, exhaust possible long tracheae section, remove excess tissue.
The digestion of 2.2 tracheal tissues and the removal of red blood cell
0.05% pronase of a little 4 DEG C of precoolings, lavation inner surface of trachea to cleaning are injected into conduit；Tracheae free-end is ligatured, after the knot at two ends confirms firmly, 0.05% pronase is filled into whole section of tracheae, and is clamped with artery clamp after the conduit of doubling, whole section of tracheae infiltration is fixed into DMEM/F12,4 DEG C are incubated overnight (about 12h).Second day, digestive juice is collected, and DMEM/F12 is drawn with 5ml syringes and rinses tracheae, flushing liquor about 5ml is collected；15ml centrifuge tubes are transferred to after digestive juice and flushing liquor are merged, and add erythrocyte cracked liquid, 1000r/min centrifugations 7min；Supernatant is removed, then with the abundant re-suspended cells of appropriate DMEM/F12.
2.3 fibroblastic removals
Re-suspended cell is inoculated in the coated 10cm culture dishes of inorganization added with the culture medium obtained by 10ml steps one.After about 1h, fibroblast is strong due to adhesive ability, shifts to an earlier date adherent compared with airway epithelial cell and is removed, the similar differential attachment method of the principle, using the coated culture dish of inorganization, is more beneficial for isolating and purifying for cell.
The original cuiture of 2.4 airway epithelial cells
After most fibroblasts are adherent, Aspirate supernatant, which is transferred in coated 24 orifice plate of step 1 collagen, to be continued to cultivate, it should now observe whether the airway epithelial cell in old culture dish is thoroughly shifted, if not having, culture medium obtained by available step one cleans transfer repeatedly, cell count is carried out again, then by every hole 5 × 104The density of individual cell is seeded into 24 orifice plates.Daily row changes liquid processing, and about 7d or so cells are up to higher degrees of fusion, and fibre swing phenomenon occur.
This law, which obtains cell, can continue the generation of Secondary Culture 3~4, but state is gradually poor, it is proposed that be tested using primary cell.
3rd, in the inspection of body physiological status
The culture plate for filling the primary airway epithelial cell of culture obtained by step 2 is moved to under model ZEISS Axiovert 200m high-power microscope observable and dynamic image is shot.For ease of the creativeness of the censorship present invention in examination as to substance, a length of 1200 milliseconds of video refers to (CD provided referring to substantive examination reference) for auditor when captured dynamic image is fabricated to by the present inventor by the interval of 40 millisecond of one pictures；30 pictures corresponding in above-mentioned video are made 30 holograms (see Fig. 1 of Figure of description) by the present inventor simultaneously.The submicroscopic structure belonged to due to single cilium on airway epithelial cell, it is difficult to observe under common high power lens, and only could produce directional swing when cell fusion degree is higher, still the bar rope shadow that is formed the rhythmic exercise of a large amount of ciliums be used as observation index.It can be observed by Fig. 1, over time, due to being influenceed by fibre swing, white bar rope shadow has corresponding change to the cell of culture.Above-mentioned dynamic change becomes apparent from the video that substantive examination reference is provided, it may be clearly seen that the bar rope shadow that " coral thicket sample " wriggles in video, this phenomenon be substantial amounts of airway epithelial cell cilium cluster caused by rhythmicity directional swing.The above results illustrate, its physiological status in body is remained using the primary airway epithelial cell of medium culture of the present invention.
Claims (2)
1. a kind of culture medium for the Primary mouse airway epithelial cell for being maintained at body physiological status, it is characterised in that every liter of culture medium is composed of the following components：DMEM/F12 culture medium 12g, penicillin G sodium salt 30mg, streptomysin 50mg, insulin 10mg, transferrins 5mg, the μ g of EGF 25, the μ g of cholera toxin 100, the μ g of hydrocortisone 108.741, ox pituitary extract 1g, bovine serum albumin(BSA) 3g, vitamin A acid 1.5 × 10-2μ g, gentamicin 50mg, amphotericin B 0.5mg, hyclone 50ml, remaining is water.
2. a kind of method for preparing culture medium described in claim 1, this method is comprised the steps of：
Penicillin, streptomysin, insulin, transferrins, EGF, cholera toxin, hydrocortisone, ox pituitary extract, bovine serum albumin(BSA), vitamin A acid, gentamicin, amphotericin B and hyclone are added in DMEM/F12 culture mediums by proportioning, add water to 1 liter of fully mixing, 0.22 μm of filtering with microporous membrane of laminar flow cell culture chamber, collects filtrate.
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| CN104263699A (en) * | 2014-09-19 | 2015-01-07 | 江苏华亿细胞组织工程有限公司 | Culture method for large-scale preparation of clinical treatment level dermal multipotent stem cells for cell transplantation |
| CN104312970A (en) * | 2014-09-19 | 2015-01-28 | 朱宁文 | Preparation method of clinical treatment level epidermal stem cell for cell therapy by applying human extracellular matrix screening and mass culture |
| CN104630134A (en) * | 2015-02-15 | 2015-05-20 | 叶中华 | Vaginal epithelial cell culture medium and vaginal epithelial cell culture method |
| CN106609258A (en) * | 2015-10-23 | 2017-05-03 | 江苏齐氏生物科技有限公司 | Rat nasal mucosa epithelial cell isolation and culture method |
| CN107312744B (en) * | 2017-06-18 | 2020-09-25 | 广东博溪生物科技有限公司 | Serum-containing oral mucosa epithelial cell culture solution |
| CN108753681B (en) * | 2018-04-28 | 2022-02-15 | 中山大学附属第一医院 | Nasal epithelial stem cell culture method and nasal epithelial stem cell proliferation culture medium |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2002088329A1 (en) * | 2001-04-30 | 2002-11-07 | North Carolina State University | Culture system for mouse tracheal epithelial cells |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2002088329A1 (en) * | 2001-04-30 | 2002-11-07 | North Carolina State University | Culture system for mouse tracheal epithelial cells |
Non-Patent Citations (4)
| Title |
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| 人与鼠神经干细胞体外培养特性的比较;祝畅 等;《中国组织工程研究与临床康复》;20070218;第11卷(第7期);第1216-1218页 * |
| 应用脱细胞气管材料建构组织工程气管的实验研究;臧梦青;《中国博士学位论文全文数据库 医药卫生科技辑 2010年第9期 E080-14》;20100915;正文第一部分 * |
| 祝畅 等.人与鼠神经干细胞体外培养特性的比较.《中国组织工程研究与临床康复》.2007,第11卷(第7期), |
| 臧梦青.应用脱细胞气管材料建构组织工程气管的实验研究.《中国博士学位论文全文数据库 医药卫生科技辑 2010年第9期 E080-14》.2010, |
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