CN102433296A - Method for culturing human airway epithelial cells - Google Patents
Method for culturing human airway epithelial cells Download PDFInfo
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Abstract
The invention discloses a method for culturing human airway epithelial cells. The method comprises the following steps of: (1) acquiring human airway epithelial cells which are subjected to primary culture; and (2) acquiring human airway epithelial cells which are subjected to subculture, namely cleaning by using a phosphoric acid buffer solution when the fusion density of primary cells reaches 70 to 90 percent, adding a 0.25 percent trypsin solution, digesting at room temperature for 5 to 10 minutes, collecting cell suspension when cells are retracted and suspended, adding an equal amount of solution containing a protease inhibitor, performing centrifugal collection on the cells, inoculating in a novel culture dish containing a complete medium at the concentration of between 1 and 6*10<6> cells/ml, culturing, and thus obtaining the human airway epithelial cells which are subjected to subculture when the cells grow to a logarithmic growth phase after 3 to 5 days. The method for culturing the human airway epithelial cells has a stable technology and is high in repeatability; and the cultured cells have high purity and uniform morphology, grow well and can be subjected to continuous passage.
Description
Technical field
The present invention is a kind of cultural method of popularity tract epithelial cell, belongs to biological technical field.
Background technology
Tracheae or segmental bronchus are the important component parts of lower respiratory tract; The airway epithelia cell is the important defensive barrier of respiratory tract; Existing discovering; Respiratory tract infection repeatedly and mucous epithelium functional disorder are closely related, and therefore, the airway epithelia cell is to seek the pathogenetic research materials of respiratory system common disease such as COPD, asthma.After the airway epithelia cell exsomatizes; Biological characteristics does not change as yet; Antimer internal state to a certain extent; And the morphological feature and the characteristics of cell biology of demonstration parent tissue, therefore the former foster foundation with cell strain of being commissioned to train of external airway epithelia cell is the effective means of studying parent tissue biological characteristic, also is that research respiratory tract disease pathogenesis and prophylactico-therapeutic measures thereof provide research tool simultaneously.In addition; The graft of trachea technology of China is in the experimental study stage always; Tissue engineering trachea is the important means of graft of trachea; And the airway epithelia cell is an indispensable part in the tissue engineering trachea as seed cell, therefore find a kind of easy, the airway epithelia cultural method is to research respiratory system disease pathogenesis and carry out graft of trachea and all have great importance efficiently.
At present; The cultural method of airway epithelia cell mainly contains: pancreatin and/or protease digestion combine mechanically peel mucous membrane method and/or mechanical wiper method; All there is following shortcoming in these methods: (1) mechanically peel mucous membrane method/mechanical wiper method is to adopt apparatus such as tweezers on tracheae or segmental bronchus, to carry out passivity to separate, and its operation easier is big, and in stripping process damage epithelial cell easily; The survivaling cell that obtains is few, and cell viability is poor.(2) trysinization/protease digestion method is to adopt pancreatin/protein enzyme solution that tracheae or segmental bronchus are carried out digestion process; To obtain epithelial cell; But the eupepsy of pancreatin/proteolytic enzyme is very strong, and the difficult grasp of digestion time, is prone to epithelial cell is caused damage; The cell pick-up rate is few, and cell survival rate is low and vigor is poor.In addition; Remain that to cause with the popularity tract epithelial cell in following reason be that the cell cultures in source is restricted: 1. lungs are open internal organs; Therefore the air flue that obtains is prone to pollute in the process of carrying out cell cultures, and the while is contained the difficulty that dregs such as a large amount of Saliva Orthanas have increased cellular segregation owing to patient airway; 2. can be used for separating the sample of airway epithelia cell; It all is the position of the tool focus of getting off from excision; The sample limited amount, and the normal tracheae in the sample or segmental bronchus are seldom, as the airway epithelia cell culture processes adopting; The pair cell damage is big, the low cell of adding of pick-up rate attaches ability, and the cell quantity that becomes to live at last is few; 3. former being commissioned to train of airway epithelia cell supported, and introduces inoblast the most easily and pollutes, and fibroblastic growth is fast, vigor good, has growth vigor, finally cause former generation airway epithelia cell cultures failure.
Summary of the invention
The cultural method that the purpose of this invention is to provide a kind of efficient, easy, highly purified popularity tract epithelial cell.
The object of the invention is realized through following technical scheme: a kind of cultural method of popularity tract epithelial cell may further comprise the steps:
(1) obtains former popularity tract epithelial cell of being commissioned to train foster: get exsomatize people's tracheae or segmental bronchus; Behind 4 ℃ of digestion 24～48h, collect the airway epithelia cell with digest medium; Be seeded to then in the petridish, carry out primary cell culture, obtain former popularity tract epithelial cell of being commissioned to train foster with perfect medium;
(2) obtain the popularity tract epithelial cell that goes down to posterity and cultivate:
After primary cell reaches 70～90% fusion density, clean with phosphoric acid buffer (PBS) solution, add 0.25% trypsin solution; Room temperature digestion 5～10 minutes; When retraction, suspension appearred in cell, collecting cell suspension-s added the equivalent solution that contains proteinase inhibitor; Centrifugal collecting cell is then with 1～6 * 10
6The concentration of cell/ml is inoculated in the new petridish that contains perfect medium and cultivates, and cell grew to logarithmic phase in 3～5 days, obtains the popularity tract epithelial cell that goes down to posterity and cultivate.
Contain 0.02% YD 30 (EDTA) in the trypsin solution in the said step (2); In petridish, the consumption of the trypsin solution of every 100mm single orifice plate is 1ml.
Proteinase inhibitor is Trypsin inhibitor SBTI (Soybean Trypsin Inhibitor) in the said step (2), and concentration is 0.5～1mg/ml.
Obtaining the former concrete steps of supporting the popularity tract epithelial cell of being commissioned to train in the said step (1) is:
1>separates all extra reticular tissue on reject tracheae or the segmental bronchus at microscopically, be cut into the segment tissue block then, with the saline water cleaning of precooling; Be immersed in the immersion substratum separating clean tracheae or segmental bronchus, behind vortex concussion 30～60s, soak 5～10min; Then rinse well, place washed tissue block in the centrifuge tube, add digest medium with the washing substratum; 4 ℃, on shaking table, digest tracheae or the piece 24～48h of bronchial tissue with rotating speed 50～60r/min;
2>tracheae or the bronchial tissue's piece that take out warp digestion are poured in the petridish, and adding foetal calf serum to final volume concentration is 10～20%, vertically cuts off tracheae; Gently scrape internal surface with cytobrush, with phosphoric acid buffer flushing internal surface and cytobrush, collect the solution that contains cast-off cells then; Behind 4 ℃ of centrifugal 5min; Clean cell with the washing substratum, behind 4 ℃ of centrifugal 5min, use the perfect medium re-suspended cell;
3>Adjust cell density with perfect medium, with 1～6 * 10
5The density of cell/ml is inoculated in the culture plate, in 37 ℃, 5%CO
2Leave standstill in the incubator and cultivated 2～3 days, obtain former popularity tract epithelial cell of being commissioned to train foster.
Step 1 of the present invention>described in the consumption of immersion substratum, washing substratum and Digestive system not have tracheae or bronchial tissue to be advisable.Described vortex concussion condition is 1000～1200r/min, and the length of described segment tissue block is 5～10cm.
Perfect medium of the present invention is the BEGM substratum that contains 95～105U/ml penicillium mould and 95～105U/ml Streptomycin sulphate.
Washing substratum of the present invention is the MEM substratum (minima essential medium) that contains 95～105U/ml penicillium mould and 95～105U/ml Streptomycin sulphate.
Immersion substratum of the present invention is that (Dithiothreitol is DTT) with 0.5～1 * 10 for the dithiothreitol dithio that contains 95～105U/ml penicillium mould, 95～105U/ml Streptomycin sulphate, 0.5～1mg/ml
-2The MEM substratum of the dnase-of mg/ml (DNase).
Digest medium of the present invention is that to contain 95～105U/ml penicillium mould, 95～105U/ml Streptomycin sulphate, final concentration be the XIV type proteolytic enzyme (ProteaseXIV) and 0.5～1 * 10 of 0.5～1mg/ml
-2The MEM substratum of the dnase-of mg/ml (DNase).
The petridish that adopts in the step according to the invention (2) is the common Tissue Culture Dish that has encapsulated 0.05～0.1% IV collagen type (Collagen type IV).The concrete making method of this petridish is:
1. the preparation of 10 * Collagen type IV: add 20ul acetate in the 10ml ultrapure water, mixing adds 0.05～0.1mg Collagen type IV;
2. petridish preparation: the 100mm petridish adds 10 * Collagen type IV, 1.5～2.5ml, shakes up, and it is tiled on the petridish, shakes up per half a hour once afterwards, inhales after two hours and abandons remaining liq, and is air-dry in super clean bench, the ultraviolet sterilization.
The present invention has following advantage compared with prior art:
1. cultural method of the present invention is consistent, good reproducibility, and cultured cells purity is high, the form homogeneous, well-grown, and can continuous passage.
2. the present invention is under the microscope direct-view, optionally removes the unnecessary tissue on tracheae or segmental bronchus surface, effectively is reduced to fibrocellular pollution and avoids damaging the airway epithelia cell; Adopt gentle reductive agent DTT and dnase-to remove tracheae or segmental bronchus surface Saliva Orthana and nucleic acid; Adopt the slowly digestion under low temperature environment of the specific Digestive system that contains XIV type proteolytic enzyme; Contain minimum medium in the Digestive system; In digestion, guarantee cell activity, and the Digestive system reaction temperature with, only digest intercellular substance and can cell membrane not cause damage; Cell attachment rate and surviving rate are significantly improved, and cultivating and can obtaining purity in 2～3 days is the airway epithelia cell more than 95%.In addition, airway epithelia is a pseudostratified ciliated columnar epithelium, and a confluent monolayer cells is only arranged, and can guarantee the quality and quantity of primary cultured cell to greatest extent with this method.
3. be coated with collagen protein IV in the petridish that uses in the cultural method of the present invention; Epithelial cell is grown on such petridish; Improved epithelial adherent property effectively; Generally, just can be almost completely adherent at the about 6h epithelium posterius cell of cultivation, solid foundation has been established in the foster success of being commissioned to train for the airway epithelia cell is former.
4. the cell that method provided by the invention obtains observes through inverted microscope and the evaluation of cellular immunization group has the form and the characteristics of typical airway epithelia cell; For the pathogenesis of further studying respiratory system diseases such as COPD, asthma has been established favourable experiment basis; And as the seed cell of tissue engineering trachea, for organizational project provides sufficient cell source.
Description of drawings
Fig. 1 is the human bronchial epithelial cell behind the 6h under the common inverted microscope (100 *).
Fig. 2 is the human bronchial epithelial cell behind the 12h under the common inverted microscope (100 *).
Fig. 3 is the human bronchial epithelial cell of common inverted microscope (100 *) after following 2 days.
Fig. 4 is the human bronchial epithelial cell of common inverted microscope (400 *) after following 3 days.
Fig. 5 is the human bronchial epithelial cell of common inverted microscope (100 *) after following 5 days.
Fig. 6 is the back 1 day human bronchial epithelial cell that goes down to posterity under the common inverted microscope (100 *).
Fig. 7 is a s-generation human bronchial epithelial cell under the common inverted microscope (100 *).
Fig. 8 a is that the immunohistochemical methods method is identified s-generation human bronchial epithelial cell, with the dyeing of Keratin sulfate immunocyte, and the DAB colour developing, tenuigenin is pale brown look positive reaction.
Fig. 8 b is the s-generation human bronchial epithelial cell as negative control that the immunohistochemical methods method is identified.
Specific embodiment
Below in conjunction with instance and accompanying drawing the present invention is described in further detail; But embodiment of the present invention is not limited in this; According to foregoing of the present invention; According to the ordinary skill knowledge and the conventional techniques means of this area, under the prerequisite that does not break away from the above-mentioned basic fundamental thought of the present invention, can also make derivation, replacement or the change of other various ways.
Embodiment 1
1. main experiment material
(1) cell source:, get the far-end lung tissue sample of the stripped lobe of the lung of excision gained because of the operation of disease row lobectomy of lungs such as lung cancer.
(2) Streptomycin sulphate of the penicillium mould of washing culture medium solution: 95U/ml, 95U/ml adds MEM substratum (minima essential medium).
(3) soaking substratum is that the adding final concentration is the dithiothreitol dithio and 0.5 * 10 of 0.5mg/ml in the washing substratum
-2The dnase-of mg/ml.
(4) digest medium is that the adding final concentration is the XIV type proteolytic enzyme and 0.5 * 10 of 0.5mg/ml in the washing substratum
-2The dnase-of mg/mL.
(5) perfect medium: the BEGM substratum that contains 95U/ml penicillium mould and 95U/ml Streptomycin sulphate.
(6) encapsulate the preparation of the petridish of 0.05%Collagen type IV
1. the acetate that adds 20ul in the ultrapure water of the preparation of 10 * Collagen type IV: 10ml, mixing, the Collagen type IV of adding 0.05mg.
2. the preparation of petridish: add 10 * Collagen type IV of 1.5ml at the common Tissue Culture Dish of 100mm, shake up, it is tiled on the petridish; Shake up per half a hour once afterwards; Inhale after two hours and abandon remaining liq, air-dry in super clean bench, the ultraviolet sterilization.
More than all solution of (1)～(5) prepare through 0.22 μ m membrane filtration degerming.
2. the cultural method of human bronchial epithelial cell
2.1 obtain human bronchial
1) sample is placed under the stereoscopic microscope, separate tracheae surrounding tissue with microscissors in the microscopically passivity with microforceps, in this process, need repeatedly with normal saline flushing so that get a clear view along the tracheae anatomical structure;
2) rinsing to visual inspection bronchial wall is totally smooth repeatedly will to separate the saline water of putting into precooling after the segmental bronchus that obtains is cut; Heighten microscopical magnification; Use microforceps and microscissors carefully to peel off residual fat and fibrous tissue adventitia outside the segmental bronchus in microscopically, wash repeatedly with the washing substratum then and remove unnecessary reticular tissue;
2.2 obtain the former foster human bronchial epithelial cell of being commissioned to train
1) segmental bronchus is cut into the segment tissue block of 5cm, cleans, be immersed in and soak in the substratum with two antibiosis reason salt solution of precooling; Soaking substratum did not have tissue to get final product; 1000r/min vortex concussion 30s soaks 5min more then, then washes tracheae three times with the washing substratum;
2) washed tissue block is put into the taper centrifuge tube of 50ml, is added digest medium to not having tissue, 4 ℃, on shaking table with rotating speed 60r/min digestion tracheae 24h;
3) will digest good tissue and Digestive system and pour in the 100mm petridish, add foetal calf serum to final volume concentration be 10% with in XIV type proteolytic enzyme.Vertically cut off tracheae, gently scrape internal surface, use phosphoric acid buffer (PBS) flushing internal surface and cytobrush then, collect the solution that contains cast-off cells with the taper centrifuge tube of 50ml with cytobrush.500g behind 4 ℃ of centrifugal 5min, abandons supernatant, cleans cell with the washing substratum, and 500g behind 4 ℃ of centrifugal 5min, uses the perfect medium re-suspended cell, cell counting;
4) second day, the overwhelming majority was unicellular adherent, and cell mass is floating, collects old perfect medium with the 50ml centrifuge tube, 500g, 4 ℃ of centrifugal 5min.What then with PBS flushing centrifuge tube once, add the 10ml prepared fresh again contains 2mM EDTA, 0.5mg/mL DTT, 0.25mg/ml collagenase (collagenase); The washing substratum of 10ug/ml DNase, 37 ℃ of incubation 15min add foetal calf serum to 10% (V/V) then; 500g, 4 ℃ of centrifugal 5min abandon supernatant; Counting, the adjustment cell density is with 1 * 10
5The density of cell/ml is planted in culture plate, places 37 ℃, 5%CO
2Leave standstill cultivation in the incubator.
2.3 obtain the human bronchial epithelial cell that goes down to posterity and cultivate
After the primary cell stand density reaches 70%, can go down to posterity.When the time comes, clean cell 3 times with 10mlPBS solution, the trypsin solution room temperature that adding 1ml 0.25% (m/v) contains 0.02%EDTA digested 5 minutes; When a large amount of suspension cell occurring; Add the equivalent solution that contains the 0.5mg/ml Trypsin inhibitor SBTI, centrifugal collecting cell is then with 1 * 10
6The concentration of cell/ml is inoculated in the new petridish that contains perfect medium and cultivates, and cell grew to logarithmic phase in 5 days, obtains the human bronchial airway epithelia cell that goes down to posterity and cultivate.
3. cultivation results
3.1 inverted phase contrast microscope is observed the former tract epithelial cell of fostering the spirit of nobility of being commissioned to train down: rounded, bright, the single uniform distribution of cell of just having planted.Visible most cell attachments about the former human bronchial epithelial cell growth 6h that is commissioned to train foster.Major part is the polygon cell, is the paving stone shape, also has less round cell and the bigger round cell (Fig. 1 and 2) of little volume of some volumes.Cell has convergence after 2 days, is polygon and inlays arrangement, and elongated spindle cell is radial or vortex shape distributes, and mostly karyon is oval; Most cells are monokaryon, and few cell has double-core or three nuclears, and nuclear is big placed in the middle; Tenuigenin is transparent, and no particle, iuntercellular are arranged closely (Fig. 3).The cell quantity showed increased is characteristic of concentration growth after 3 days, and visible elongated spindle cell and paving stone like cell mix mutually, and projection link to each other (Fig. 4) is often arranged between grown cell.Cell growth in 5 days reaches logarithmic phase growth, subregion cell overlap accumulated growth (Fig. 5).
Cultivate the airway epithelia cell 3.2 inverted phase contrast microscope is observed down to go down to posterity: the cell speed of growth is obviously accelerated than primary cell.Going down to posterity was grown to monolayer in back 3 days, and form is similar with former generation, but cell volume increases to some extent, and elongated (Fig. 6 and 7) gradually.
3.3 cellular immunization group evaluation: carry out immunohistochemical methods with mouse-anti people cytokeratin (AE1/AE3) monoclonal antibody and identify; Tenuigenin is pale brown look; Nucleus is that (Fig. 8 a) for mazarine; Do not add simultaneously mouse-anti people cytokeratin monoclonal antibody and carry out the immunohistochemical methods evaluation as negative control, an only visible nucleus is mazarine (Fig. 8 b).The ultra quick UItraSensitve of instant immunohistochemistry that concrete steps are produced referring to Fuzhou Maixin biotechnology Development Co., Ltd
TMSP test kit (KIT-9710) specification sheets carries out.The positive painted cell count of counting under light microscopic.Purity (%)=positive painted cell count/TCS * 100% repeats 3 times and averages.5 visuals field of every sheet picked at random, 200 cells are detected in each visual field at least.Calculate cell purity in each visual field, average out to is more than 95%.
Embodiment 2
1. main experiment material
(1) cell source:, get the far-end lung tissue sample of the stripped lobe of the lung of excision gained because of the operation of disease row lobectomy of lungs such as lung cancer.
(2) Streptomycin sulphate of the penicillium mould of washing culture medium solution: 105U/ml, 105U/ml adds the MEM substratum.
(3) soaking substratum is that the adding final concentration is the dithiothreitol dithio and 1 * 10 of 1mg/ml in the washing substratum
-2The dnase-of mg/ml.
(4) digest medium is that the adding final concentration is the XIV type proteolytic enzyme and 1 * 10 of 1mg/ml in the washing substratum
-2The dnase-of mg/mL.
(5) perfect medium: the BEGM substratum that contains 105U/ml penicillium mould and 105U/ml Streptomycin sulphate.
(6) encapsulate the preparation of the petridish of 0.1%Collagen type IV
1. the acetate that adds 20ul in the ultrapure water of the preparation of 10 * Collagen type IV: 10ml, mixing, the Collagen type IV of adding 0.1mg.
2. the preparation of petridish: add 10 * Collagen type IV of 2.5ml at the common Tissue Culture Dish of 100mm, shake up, it is tiled on the petridish; Shake up per half a hour once afterwards; Inhale after two hours and abandon remaining liq, air-dry in super clean bench, the ultraviolet sterilization.
More than all solution of (1)～(5) prepare through 0.22 μ m membrane filtration degerming.
2. the cultural method of human bronchial epithelial cell
2.1 obtain human bronchial
1) sample is placed under the stereoscopic microscope, separate segmental bronchus surrounding tissue with microscissors in the microscopically passivity with microforceps, in this process, need repeatedly with normal saline flushing so that get a clear view along the tracheae anatomical structure.
2) rinsing to visual inspection bronchial wall is totally smooth repeatedly will to separate the saline water of putting into precooling after the segmental bronchus that obtains is cut; Heighten microscopical magnification; Use microforceps and microscissors carefully to peel off residual fat and fibrous tissue adventitia outside the segmental bronchus in microscopically, wash repeatedly with the washing substratum then and remove unnecessary reticular tissue.
2.2 obtain the former foster human bronchial epithelial cell of being commissioned to train
1) segmental bronchus is cut into the segment tissue block of 10cm, cleans, be immersed in and soak in the substratum with two antibiosis reason salt solution of precooling; Soaking substratum did not have tissue to get final product; 1200r/min vortex concussion 60s soaks 5min more then, then washes segmental bronchus three times with the washing substratum;
2) washed tissue block is put into the taper centrifuge tube of 50ml, is added digest medium to not having tissue, 4 ℃, on shaking table with rotating speed 50r/min digestion tracheae 36h;
3) will digest good tissue and Digestive system and pour in the 100mm petridish, add foetal calf serum to final volume concentration be 20% with in XIV type proteolytic enzyme.Vertically cut off tracheae, gently scrape internal surface,, collect the solution that contains cast-off cells with the taper centrifuge tube of 50ml then with phosphoric acid buffer PBS flushing internal surface and cytobrush with cytobrush.500g behind 4 ℃ of centrifugal 5min, abandons supernatant, cleans cell with the washing substratum, and 500g behind 4 ℃ of centrifugal 5min, uses the perfect medium re-suspended cell, cell counting;
4) second day, the overwhelming majority was unicellular adherent, and cell mass is floating, collects old perfect medium with the 50ml centrifuge tube, 500g, 4 ℃ of centrifugal 5min.What then with PBS flushing centrifuge tube once, add the 10ml prepared fresh again contains 2mM EDTA, 0.5mg/mLDTT, 0.25mg/ml collagenase (collagenase), the washing substratum of 10ug/ml DNase, 37 ℃ of incubation 1h.Add foetal calf serum to 10% (V/V) then, 500g, 4 ℃ of centrifugal 5min abandon supernatant, counting, the adjustment cell density is with 1 * 10
5The density of cell/ml is planted in culture plate, places 37 ℃, 5%CO
2Leave standstill cultivation in the incubator.
2.3 obtain the human bronchial epithelial cell that goes down to posterity and cultivate
After the primary cell stand density reaches 90%, can go down to posterity.When the time comes, clean cell 3 times with 10mlPBS solution, the trypsin solution room temperature that adding 1ml 0.25% (m/v) contains 0.02%EDTA digested 10 minutes; When a large amount of suspension cell occurring; Add and contain 1mg/ml Trypsin inhibitor SBTI equivalent solution, centrifugal collecting cell is then with 6 * 10
6The concentration of cell/ml is inoculated in the new petridish that contains perfect medium and cultivates, and cell grew to logarithmic phase in 3 days, obtains the human bronchial airway epithelia cell that goes down to posterity and cultivate.
3. cultivation results
The result is identical with embodiment one.
Embodiment 3
1. main experiment material
(1) cell source:, get the stripped lung tissue sample of excision gained because of serious capable lung transplants of pulmonary disorder such as pulmonary emphysema.
(2) Streptomycin sulphate of the penicillium mould of washing culture medium solution: 105U/ml and 105U/ml adds the MEM substratum.
(3) soaking substratum is that the adding final concentration is the dithiothreitol dithio and 1 * 10 of 1mg/ml in the washing substratum
-2The dnase-of/ml.
(4) digest medium is that the adding final concentration is the XIV type proteolytic enzyme and 1 * 10 of 1mg/ml in the washing substratum
-2The dnase-of mg/mL.
(5) perfect medium: the BEGM substratum that contains 105U/ml penicillium mould and 105U/ml Streptomycin sulphate.
(6) encapsulate the preparation of the petridish of 0.1%Collagen type IV
1. the acetate that adds 20ul in the ultrapure water of the preparation of 10 * Collagen type IV: 10ml, mixing, the Collagen type IV of adding 0.1mg.
2. the preparation of petridish: add 10 * Collagen type IV of 2.5ml at the common Tissue Culture Dish of 100mm, shake up, it is tiled on the petridish; Shake up per half a hour once afterwards; Inhale after two hours and abandon remaining liq, air-dry in super clean bench, the ultraviolet sterilization.
More than all solution of (1)-(5) prepare through 0.22 μ m membrane filtration degerming.
2. the cultural method of human bronchial epithelial cell
2.1 obtain people's tracheae
1) sample is placed under the stereoscopic microscope, separate the tracheae surrounding tissue with microforceps with microscissors quick passivity under mirror, in this process, need repeatedly with normal saline flushing so that get a clear view along the tracheae anatomical structure;
2) rinsing to visual inspection tracheal wall is totally smooth repeatedly will to separate the saline water of putting into precooling after the tracheae that obtains is cut; Heighten microscopical magnification; Use microforceps and microscissors carefully to peel off residual fat and fibrous tissue adventitia outside the tracheae in microscopically, wash repeatedly with the washing substratum then and remove unnecessary reticular tissue;
2.2 obtain former people's tracheae epithelial cell of being commissioned to train foster
1) tracheae is cut into the segment tissue block of 5cm, cleans, be immersed in and soak in the substratum with two antibiosis reason salt solution of precooling; Soaking substratum did not have tissue to get final product; 1200r/min vortex concussion 60s soaks 5min more then, then washes tracheae three times with the washing substratum;
2) washed tissue block is put into the taper centrifuge tube of 50ml, is added digest medium to not having tissue, 4 ℃, on shaking table with rotating speed 55r/min digestion tracheae 48h;
3) will digest good tissue and Digestive system and pour in the 100mm petridish, add foetal calf serum to final volume concentration be 20% with in XIV type proteolytic enzyme.Vertically cut off tracheae, gently scrape internal surface,, collect the solution that contains cast-off cells with the taper centrifuge tube of 50ml then with phosphoric acid buffer PBS flushing internal surface and cytobrush with cytobrush.500g behind 4 ℃ of centrifugal 5min, abandons supernatant, cleans cell with the washing substratum, and 500g behind 4 ℃ of centrifugal 5min, uses the perfect medium re-suspended cell, cell counting;
4) second day, the overwhelming majority was unicellular adherent, and cell mass is floating, collects old perfect medium with the 50ml centrifuge tube, 500g, 4 ℃ of centrifugal 5min.What then with PBS flushing centrifuge tube once, add the 10ml prepared fresh again contains 2mM EDTA, 0.5mg/mLDTT, 0.25mg/ml collagenase, the washing substratum of 10ug/ml DNase, 37 ℃ of incubation 1h.Add foetal calf serum to 10% (V/V) then, 500g, 4 ℃ of centrifugal 5min abandon supernatant, counting, the adjustment cell density is with 1 * 10
5The density of cell/ml is planted in culture plate, places 37 ℃, 5%CO
2Leave standstill cultivation in the incubator.
2.3 obtain the people's tracheae epithelial cell that goes down to posterity and cultivate
After the primary cell stand density reaches 80%, can go down to posterity.When the time comes, clean cell 3 times with 10mlPBS solution, the trypsin solution room temperature that adding 1ml 0.25% (m/v) contains 0.02%EDTA digested 10 minutes; When a large amount of suspension cell occurring; Add the equivalent solution that contains the 1mg/ml Trypsin inhibitor SBTI, centrifugal collecting cell is then with 6 * 10
6The concentration of cell/ml is inoculated in the new petridish that contains perfect medium and cultivates, and cell grew to logarithmic phase in 3 days, obtains the people's tracheae airway epithelia cell that goes down to posterity and cultivate.
3. cultivation results
The result is identical with embodiment one.
Claims (10)
1. the cultural method of a popularity tract epithelial cell is characterized in that, may further comprise the steps:
(1) obtains former popularity tract epithelial cell of being commissioned to train foster: get exsomatize people's tracheae or segmental bronchus; Behind 4 ℃ of digestion 24～48h, collect the airway epithelia cell with digest medium; Be seeded to then in the petridish, carry out primary cell culture, obtain former popularity tract epithelial cell of being commissioned to train foster with perfect medium;
(2) obtain the popularity tract epithelial cell that goes down to posterity and cultivate:
After primary cell reaches 70～90% fusion density, clean with the phosphoric acid buffer liquor, add 0.25% trypsin solution; Room temperature digestion 5～10 minutes; When retraction, suspension appearred in cell, collecting cell suspension-s added the equivalent solution that contains proteinase inhibitor; Centrifugal collecting cell is then with 1～6 * 10
6The concentration of cell/ml is inoculated in the new petridish that contains perfect medium and cultivates, and cell grew to logarithmic phase in 3～5 days, obtains the popularity tract epithelial cell that goes down to posterity and cultivate.
2. the cultural method of popularity tract epithelial cell according to claim 1 is characterized in that, contains 0.02% YD 30 in the trypsin solution in the said step (2); In petridish, the consumption of the trypsin solution of every 100mm single orifice plate is 1ml.
3. the cultural method of popularity tract epithelial cell according to claim 1 is characterized in that, proteinase inhibitor is a Trypsin inhibitor SBTI in the said step (2), and concentration is 0.5～1mg/ml.
4. the cultural method of popularity tract epithelial cell according to claim 1 is characterized in that, obtains the former concrete steps of supporting the popularity tract epithelial cell of being commissioned to train in the said step (1) to be:
1>separates all extra reticular tissue on reject tracheae or the segmental bronchus at microscopically, be cut into the segment tissue block then, with the saline water cleaning of precooling; Be immersed in the immersion substratum separating clean tracheae or segmental bronchus, behind vortex concussion 30～60S, soak 5～10min; Then rinse well, place washed tissue block in the centrifuge tube, add digest medium with the washing substratum; 4 ℃, on shaking table, digest tracheae or the piece 24～48h of bronchial tissue with rotating speed 50～60r/min;
2>tracheae or the bronchial tissue's piece that take out warp digestion are poured in the petridish, and adding foetal calf serum to final volume concentration is 10～20%, vertically cuts off tracheae; Gently scrape internal surface with cytobrush, with phosphoric acid buffer flushing internal surface and cytobrush, collect the solution that contains cast-off cells then; Behind 4 ℃ of centrifugal 5min; Clean cell with the washing substratum, behind 4 ℃ of centrifugal 5min, use the perfect medium re-suspended cell;
3>Adjust cell density with perfect medium, with 1～6 * 10
5The density of cell/ml is inoculated in the culture plate, in 37 ℃, 5%CO
2Leave standstill in the incubator and cultivated 2～3 days, obtain former popularity tract epithelial cell of being commissioned to train foster.
5. the cultural method of popularity tract epithelial cell according to claim 1 is characterized in that, described step 1>described in immersion substratum, the consumption of washing substratum and Digestive system to not have tracheae or bronchial tissue; Described vortex concussion condition is 1000～1200r/min, and the length of described segment tissue block is 5～10cm.
6. the cultural method of popularity tract epithelial cell according to claim 1 is characterized in that, described perfect medium is the BEGM substratum that contains 95～105U/ml penicillium mould and 95～105U/ml Streptomycin sulphate.
7. the cultural method of popularity tract epithelial cell according to claim 1 is characterized in that, described washing substratum is the MEM substratum that contains 95～105U/ml penicillium mould and 95～105U/ml Streptomycin sulphate.
8. the cultural method of popularity tract epithelial cell according to claim 1 is characterized in that, described immersion substratum is the dithiothreitol dithio and 0.5～1 * 10 that contains 95～105U/ml penicillium mould, 95～105U/ml Streptomycin sulphate, 0.5～1mg/ml
-2The MEM substratum of the dnase-of mg/ml.
9. the cultural method of popularity tract epithelial cell according to claim 1 is characterized in that, described digest medium is that to contain 95～105U/ml penicillium mould, 95～105U/ml Streptomycin sulphate, final concentration be the XIV type proteolytic enzyme and 0.5～1 * 10 of 0.5～1mg/ml
-2The MEM substratum of the dnase-of mg/ml.
10. the cultural method of popularity tract epithelial cell according to claim 1 is characterized in that, the petridish that adopts in the said step (2) is the common Tissue Culture Dish that has encapsulated 0.05～0.1% IV collagen type.
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| CN102787095A (en) * | 2012-07-06 | 2012-11-21 | 广州医学院第一附属医院 | Method for cultivating rat primary airway epithelial cells |
| CN105274050A (en) * | 2015-11-12 | 2016-01-27 | 常州大学 | Culture method of human airway epithelial cells for treating bronchial asthma |
| CN105441379A (en) * | 2015-12-24 | 2016-03-30 | 李童斐 | Method for culturing airway epithelial cells by simply, conveniently and rapidly inducing differentiation of cilia |
| CN105820996A (en) * | 2016-04-18 | 2016-08-03 | 浙江大学 | Human primary airway epithelial cell culture method |
| CN107129965A (en) * | 2017-06-07 | 2017-09-05 | 新乡医学院 | Human bronchial epithelial cell differentiation culture and authentication method |
| CN107236700A (en) * | 2017-06-12 | 2017-10-10 | 新乡医学院 | The culture of bronchus primary epithelial cells and authentication method |
| CN110484488A (en) * | 2019-08-29 | 2019-11-22 | 浙江大学医学院附属第一医院 | The method for building up of people's throat leukoplakia epithelial cell dimensional culture model and its application |
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