CN102391984A - In-vitro culture method for embryo lungs - Google Patents
In-vitro culture method for embryo lungs Download PDFInfo
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- CN102391984A CN102391984A CN2011103520894A CN201110352089A CN102391984A CN 102391984 A CN102391984 A CN 102391984A CN 2011103520894 A CN2011103520894 A CN 2011103520894A CN 201110352089 A CN201110352089 A CN 201110352089A CN 102391984 A CN102391984 A CN 102391984A
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Abstract
The invention belongs to the biotechnology field, relates to an in-vitro organ culture method for embryo lungs of mammals, and particularly relates to the isolated organ culture for the lungs in embryonic periods of mice. The in-vitro organ culture method solves the main technical problems that a conventional culture method has the disadvantages of needing special materials and occupying large space. The in-vitro organ culture method for the embryo lungs adopts the technical scheme that the method comprises the steps as follows: carrying tablets are put in cultivation holes formed on a porous culture plate and are matched with the cultivation holes in size, and a cultivation space is formed at the bottom part of each cultivation hole; secondly, the obtained mammalian embryo lungs are put on the carrying tablets, culture mediums are added in, and the lungs are led to be positioned at the junctions of the culture mediums and air; and thirdly, the lungs are cultured in an incubator under the temperature of 37 DEG C, and then solution changing of the culture mediums is carried out every one day or every two days. By adopting the in-vitro organ culture method, large number of in vitro cultured lung organs that are grown stably and are convenient for subsequent operation can be obtained at low cost, and the requirements of large-scale drug screening can be satisfied.
Description
Technical field
The invention belongs to biological technical field, be specifically related to the external organ culture method of mammiferous embryo lung, particularly the isolated organ of mice embryonic phase lung is cultivated.
Background technology
Mammiferous allelotaxis's research can be understood a plurality of basic cytobiology processes under the background of complete organ.This is in genetic epoch particularly important, because genetically deficient in Mammals such as the mouse etc. or sudden change can directly be associated with human congenital malformation.
External organ culture belongs to a kind of advanced form of tissue culture; It can reproduce the growth course of organ; The functional status of simulated organ under different states and condition is to understand allelotaxis's process, the important technical of study of disease mechanism of causing a disease and drug screening.This is particularly useful for the organ lung of cultivating through the lateral configuration mechanism.The growth of mice embryonic lung starts from the evagination of 9.5 days preceding gut entoderm laryngotracheal groove of embryo, forms bastem then and forms bronchial tree gradually.At this time each bastem is comprising three layers, epidermis, and a mesenchyme that holds and a cortex form the lung of respiratory system at last.And these branches, the organ formation of layering and lung needs the for example regulation and control of Urogastron (EGF) fibroblast growth factor cytokines such as (FGF) and signal path.Influential in order to confirm these processes of which factor pair, the vitro culture experimental system of body early embryo lung just arises at the historic moment.
The vitro culture system of at present more common embryo lung is based on Trowell nineteen fifty-nine The culture of mature organs in a synthetic medium.Exptl.CeZZ Res.16:118-147; 1959. many thereafter people improve it; But its core is for utilizing heavy caliber petridish (35mm; 60mm), add an amount of substratum, the polycarbonate filter membrane about the 5um of aperture is floated on the substratum.Then the mice embryonic lung is positioned on the filter membrane, places the gas-liquid intersection to cultivate (Fig. 1,2,3,4).But the use of aforesaid method needs (1) to use exotic materialss such as polycarbonate filter membrane; (2) a large amount of petridish space of waste; (3) contact area of embryo lung and substratum is limited is unfavorable for that further effectively experiment is intervened, (4) and be unfavorable for the application that extensive embryo lung is cultivated.
Summary of the invention
The objective of the invention is to solve conventional cultural method and need use exotic materialss such as polycarbonate filter membrane, waste a large amount of problems such as petridish space.
This programme solution provides a kind of external organ culture method of mammal embryo lung, it is characterized in that may further comprise the steps:
A, in the culture hole of porous culture plate, put into the adaptive with it carrying tablet of size, construct culture space in the culture hole bottom;
B, the mammal embryo lung of obtaining is positioned on the carrying tablet, adds substratum, make embryo lung be in the liquid-gas interface of substratum and air;
Cultivate in c, 36 ℃～38 ℃ incubators, carried out substratum and change liquid in per then 1 to 2 day.
Wherein, the described porous culture plate of aforesaid method step a is 24 well culture plates (arrangement of 4X 6 holes, bore dia 15.6mm, the hole floorage 1.9m of standard
2).
Wherein, the described porous culture plate of aforesaid method step a is merely the carrying container of liquid nutrient medium, does not directly contact with embryo lung, can use standard 24 well culture plates of home-made sterilization.
Wherein, the carrying tablet described in the aforesaid method step a is the transparent glass disk.Directly contact with the embryo lung of cultivating, should carry out corresponding cytobiology and handle.The size of carrying tablet is slightly less than culture hole hole floorage, gets final product for the 50-80% of culture hole is big or small.
Wherein, add moisture equilibrium liquid at each interporal lacuna among the aforesaid method step a, to slow down the substratum volatilization at the porous culture plate that uses.
Wherein, the liquid of moisture equilibrium described in the aforesaid method is aseptic phosphate buffered saline buffer or zero(ppm) water.
Wherein, embryo lung described in the aforesaid method step b is the mouse lung original hase of 10～13 days isolating embryonic stages.
Wherein, the lung modes of emplacement described in the aforesaid method step b is: embryo lung is kept flat.Make embryo lung be in the liquid of substratum-gas intersection.
Wherein, the adjustment carrying tablet is in the center of the bottom of culture hole behind the adding substratum among the aforesaid method step b, and the mammal embryo lung is positioned at the middle part of carrying tablet.
Wherein, The mode that substratum described in the aforesaid method c step adopts half amount to increase progressively is changed liquid; Keep the half the in former culture hole of culture volume last time when promptly changing liquid at every turn, then will last time half of the culture volume fresh culture that by volume increases 4%-16% more than the mark add culture hole.The preferred half the 8%-13% of culture volume that increases, optimum is 10%-12% more.
The beneficial effect of the inventive method is: overcome the big shortcoming of taking up room of present all methods, improved space availability ratio greatly, also reduced cost simultaneously, be convenient to large scale culturing.Because lung is more with contacting of substratum, improved the efficient that experiment is intervened.Small volume is cultivated the consumption that has reduced substratum, only needs the culture medium culturing about 180ul to get final product.Overcome prior art and need need not expensive imported materials and items, the import filter membrane takies the prejudice that big culture space and resource just can obtain effect, and cost reduces greatly.When doing the experiment intervention, also can reduce concentration or effect that the amount of intervening reagent just can reach expection.Assembling and simple to operate only needs the deckglass of handling well is put into culture hole, places embryo lung, adds culture medium culturing and gets final product, and is simple.The cultivation form that better lung is arranged owing to there be not blocking of support membrane, is convenient to observe the tracheae branch of lung, observes lung in the vitro culture situation as using general cell inverted microscope.And the deckglass that adds has also made things convenient for subsequent operations, as carrying out in situ hybridization, operations such as immunofluorescence.The inventive method extracorporeal culturing embryo lung surpasses 10 days, and lung's bronchial smooth muscle can rhythmic contractile motion, has good application prospect.
Description of drawings
The system synoptic diagram of Fig. 1, existing embryo lung cultural method.1,60mm petridish; 2, embryo lung; 3 polycarbonate leaching films; 4, substratum.
The system synoptic diagram of Fig. 2, existing embryo lung cultural method.1,60mm petridish; 2, embryo lung; 3 polycarbonate leaching films; 4, substratum.
The system synoptic diagram of Fig. 3, embryo lung cultural method of the present invention.1, the culture hole on 24 well culture plates; 2 embryo lungs; 3, substratum; 4, carrying tablet.
The system synoptic diagram of Fig. 4, embryo lung cultural method of the present invention.1, the culture hole on 24 well culture plates; 2 embryo lungs; 3, substratum; 4, carrying tablet.
Fig. 5, demonstration be that the embryos of 12.5 days mice embryonic phases cultivates ten days growing state.
Fig. 6 is presented at the 5th day that embryo lung is cultivated for video interception; After this rhythmic contractile motion appears in lung's bronchial smooth muscle; Wherein the indicated position of arrow is quite significantly position of motion amplitude, lays respectively at the big segmental bronchus of lung and two tracheae branches that the lobe of the lung is little.
Fig. 7, replacing culture volume scheme.
The picture of Fig. 8, standard 24 porocyte culture plates is to show the space that is used to add the humidity balance liquid that exists between its each hole.Surround a gap between every adjacent 4 culture hole, standard 24 porocyte culture plates have 15 gaps that can add moisture equilibrium liquid.1 is gap between culture hole among the figure, and 2 is culture hole.
The photo of Fig. 9, standard 24 porocyte culture plates is to show the space that is used to add the humidity balance liquid that exists between its each hole.Surround a gap between every adjacent 4 culture hole, standard 24 porocyte culture plates have 15 gaps that can add moisture equilibrium liquid.1 is gap between culture hole among the figure, and 2 is culture hole.
Embodiment:
, through embodiment the present invention is elaborated below in conjunction with accompanying drawing.
The invention provides a kind of external organ culture method of mammal embryo lung, may further comprise the steps:
A, in the culture hole of porous culture plate, put into the adaptive with it carrying tablet of size, construct culture space in the culture hole bottom;
B, the mammal embryo lung of obtaining is positioned on the carrying tablet, adds substratum, make embryo lung be in the liquid-gas interface of substratum and air;
Cultivate in c, 36 ℃～38 ℃ incubators, carried out substratum and change liquid in per then 1 to 2 day.
Wherein, the described porous culture plate of aforesaid method step a is 24 well culture plates (arrangement of 4X 6 holes, bore dia 15.6mm, the hole floorage 1.9m of standard
2).
Wherein, the described porous culture plate of aforesaid method step a is merely the carrying container of liquid nutrient medium, does not directly contact with embryo lung, can use standard 24 well culture plates of home-made sterilization.
Wherein, the carrying tablet described in the aforesaid method step a is the transparent glass disk, directly contacts with the embryo lung of cultivating, and must carry out corresponding general cytobiology and handle.The carrying tablet size should be less than pore size, and size is the 50-80% size of culture hole, form fit.Preferably the same with culture hole is circular transparent glass disk.Carrying tablet generally should be attached to the bottom.
Wherein, can add moisture equilibrium liquid at each interporal lacuna of the porous culture plate that uses in the aforesaid method, to slow down the substratum volatilization.
Wherein, the liquid of moisture equilibrium described in the aforesaid method is aseptic phosphate buffered saline buffer or zero(ppm) water.
Wherein, embryo lung described in the aforesaid method step b is the mice embryonic lung of 10～13 days isolating embryonic stages.
Wherein, the lung modes of emplacement described in the aforesaid method step b is: embryo lung is kept flat.Make embryo lung be in the liquid of substratum-gas interface.
Wherein, the adjustment carrying tablet is in the center of the bottom of culture hole behind the adding substratum among the aforesaid method step b, and the mammal embryo lung is positioned at the middle part of carrying tablet.
Wherein, The mode that substratum described in the aforesaid method c step adopts half amount to increase progressively is changed liquid; Keep the half the in former culture hole of culture volume last time when promptly changing liquid at every turn, then will last time half of the culture volume fresh culture that by volume increases 4%-16% more than the mark add culture hole.
All ingredients that uses in the inventive method and material all should be aseptic by this area general knowledge, and operation also should be carried out under aseptic condition.
The carrying tablet that the inventive method is used can be the thin slice that histocyte is cultivated that is suitable for of various materials, and the most frequently used and optimum is the transparent glass disk.Carrying tablet should clean and sterilising treatment before using fully, under the situation of needs, can use suitable matrix pre-treatment, like poly-lysine, and gelatin or collagen etc.
The porous culture plate that uses in the inventive method can be conventional can be applied to common 24 orifice plates that cell tissue is cultivated, its hole preferably floorage at 1～3 square centimeter flat circular hole.Modal is the flat Tissue Culture Plate in 24 holes of standard, and material is that glass or plastics all can.Standard specifications is that cloth, bore dia 15.6mm, hole floorage 1.9m are arranged in 4X 6 holes
2
The moisture equilibrium liquid that uses in the inventive method must be aseptic, nontoxic.The most handy aseptic phosphate buffered saline buffer or zero(ppm) water are according to using the porous culture plate to confirm consumption.Porous cultivates and to have the gap between plate hole that (referring to Fig. 8, Fig. 9), can be used for adding moisture equilibrium liquid in the present invention, the moisture equilibrium liquid that use in the gap between general each hole of flat Tissue Culture Plate, standard 24 holes is long-pending should be greater than 200ul, preferably 500ul.
The embryo lung source of using in the inventive method is the embryo of the mouse of general experiment usefulness.The experiment mice embryo lung can be the lung original hase (the lung original hase is a kind of call that early stage embryo lung is grown in this area, generally comprises the tracheae and the lobe of the lung that divide expenditure from oesophagus) of 10～13 days embryonic stages.And preferably only cultivate 1 mice embryonic lung on each carrying tablet.Embryo lung preferably keeps flat, and promptly is placed horizontally at carrying tablet central authorities by the square section so that the lung original hase can develop into the good form of form.And substratum is to make embryo lung be in the liquid-gas interface of substratum and air after adding; This description of this area is meant that substratum can not flood embryo lung fully; But it is directly exposed in the air; But to make its liquid film at upper surface formation one deck substratum, this just is referred to as to be in the liquid-gas interface of substratum and air.
When obtaining embryo lung, the integrity of embryo lung in must guaranteeing to perform the operation (not destroying embryo lung tracheae branch) in order to avoid the lung development paramophia occurs.
Use substratum to can be the general various cell culture mediums in this area in the inventive method; Such as dulbecco ' s modifi ed eagle ' s medium (DMEM; General substratum, like the product of the Invitrogen of businessman article No. 11965, but or also disclosed on the reference net:
Http:// baike.baidu.com/view/3501452.htmDMEM (H) cell culture medium (powder-type) of record becomes assignment system) or Eagle ' s minimal essential medium (it is known to fill a prescription for EMEM, general substratum.In the substratum of selecting, adding volume(tric)fraction then is FBS (foetal calf serum) or F12 nutritional factor (the Invitrogen Company products of 5-20%; The name of an article: nutritional factor mixture; Article No.: 21700), generally also can add by final concentration 0.1mg/ml interpolation microbiotic penicillin and streptomycin (penicillium mould and Streptomycin sulphate) preventing to pollute.
Culture condition in the inventive method is general cell culture condition.General temperature is 36 ℃～38 ℃; Be preferably under 37 ℃, volume(tric)fraction is to cultivate in the environment of 5% carbonic acid gas.
The substratum that uses in the inventive method can remove old substratum and add the new substratum of equal-volume and continue to cultivate whenever at a distance from 24～48 hours.The liquid mode of preferably changing that can also adopt is that the mode that adopts half amount to increase progressively is changed liquid; Keep the half the in former culture hole of culture volume last time when promptly changing liquid at every turn, then will than last time culture volume half also by volume the mark fresh culture that increases 4%-16% more add culture hole.The preferred half the 8%-13% of culture volume that increases, optimum is 11% more.If during with the flat Tissue Culture Plate in standard 24 holes, culture volume can be used every hole 150-200ul, best volume should be every hole 180ul.Will be when changing substratum than the half the fresh culture adding culture hole of Duo 5～15ul of culture volume last time.Preferred 8～12ul, the best is 10ul.
Embodiment one: use the inventive method to cultivate 11.5 days Balb/c mice embryonic phases lung.
1, prepares carrying tablet: as carrying tablet, buy magnificent triumphant experiment glassware ltd in Haimen City with the glass dome sheet of diameter 12mm.At first, use zero(ppm) water dose volume mark is 1% hydrochloric acid soln, shakes in room temperature and cleans the dome sheet one hour.After cleaning with zero(ppm) water, put into volume(tric)fraction and be 70% spirituous solution, shake to clean and spend the night.Hold the dome sheet after the cleaning with the 10cm glass dish, put into the high-temperature steam Autoclave after draining liquid, 121 ℃ of sterilizations 40 minutes.It is subsequent use that oven dry is placed on Bechtop.
2, assembling culture apparatus: the dome sheet of handling well with aseptic tweezers gripping is positioned in the culture hole of 24 aseptic well culture plates.24 aseptic well culture plates are for purchasing in the 3524 type tissue culturing plates of Costar (Corning Incorporated, the U.S.).
3, obtain mouse lung 11.5 days embryonic stages: with the female mouse of the Balb/c that becomes pregnant; Derive from Huaxi Hospital Attached to Sichuan Univ genetically engineered mouse center SPF level (not having special pathogenic bacteria) Animal House; Detect vaginal suppository and be designated as E0.5 days the same day and begin to calculate the fetal development time, took out pregnant mouse from the animal feeding room at E11.5 days.After the method for craning one is put to death pregnant mouse, disinfect pregnant mouse and disscting instrument in alcohol.In the dissection liquid (1X PBS, HBSS or DMEM) of precooling on ice, dissect after taking out mice embryonic, stereoscope takes out embryo lung down, transfers in the EP pipe that substratum (DMEM+10%FBS) 1.5ml is housed with the 1mL suction nozzle.
4, in Bechtop, shift embryo lung to culture hole, one in every hole with the pipettor of 1mL.Siphon away in the culture hole residual media and add the 180ul substratum with pipettor.With spirit lamp high-temperature sterilization point tweezers, treat cover plate middle part, adjustment embryo lung to garden, cold back, and adjustment cover plate position, garden makes embryo lung be positioned at the culture hole center.The structure of culture system is referring to the synoptic diagram of Fig. 3 and Fig. 4.
5, the embryo lung that carefully will plant is positioned over cell culture incubator in 37 ℃, 5%CO
2The middle cultivation 24 hours.
6, with pipettor with keeping former substratum 90ul in the culture hole, and abandon the residue substratum.
7, the fresh culture that adds 90+10ul is cultivated in cell culture incubator behind the mixing to culture hole.
8, cultivate after 24 hours, carry out half with step 6 and 7 then and measure to increase progressively and change liquid: stay the new substratum of the former substratum+105ul of 95ul.
9, every liquid that changed at a distance from 24 hours.It is as shown in Figure 7 to change the culture volume scheme.
10, cultivate 10 days effects (seeing accompanying drawing 5).
Fig. 5 shows is that the embryos of 12.5 days mice embryonic phases cultivates ten days growing state.The time of cultivating embryo lung can surpass 10 days, and sufficient branch of lung qi pipe branch and expansion, has obtained good form.
Fig. 6 is presented at the 5th day that embryo lung is cultivated for video interception; After this rhythmic contractile motion appears in lung's bronchial smooth muscle; Wherein the indicated position of arrow is quite significantly position of motion amplitude, lays respectively at the big segmental bronchus of lung and two tracheae branches that the lobe of the lung is little.Explain and cultivate kidney, certain senior physiological function is arranged near situation in the body.
Through test of many times, it almost is 100% success ratio that the embryo lung that utilizes aforesaid method to use E12.5 days carries out vitro culture, adopts the mode of 1 embryo lung in 1 hole, and the dull and stereotyped institute in 24 holes is porose cultivates 24, and the best is to use the hole of 8 inner rings to cultivate.24 orifice plates (13cmX8.5cmX2.5cm) are cultivated 24 embryo lungs.1 lung in promptly about 11 cubic centimetres of spaces; And the prior art minimum is foster one of general 3.5cmX3.5cmX2.5cm; 1 lung in promptly about 31 cubic centimetres of spaces; The cultivation of 24 lungs of this programme can be joined together as the operation of the strictness of equivalent environment control together in addition, and the growth consistence is better, also is more convenient for realizing the large-scale medicine screening.
Claims (9)
1. the external organ culture method of mammal embryo lung is characterized in that may further comprise the steps:
A, in the culture hole of porous culture plate, put into the adaptive with it carrying tablet of size, construct culture space in the culture hole bottom;
B, the mammal embryo lung of obtaining is positioned on the carrying tablet, adds substratum, make embryo lung be in the liquid-gas interface of substratum and air;
Cultivate in c, 36 ℃～38 ℃ incubators, carried out substratum and change liquid in per then 1 to 2 day.
2. the external organ culture method of mammal embryo lung according to claim 1 is characterized in that: the described porous culture plate of step a is 24 well culture plates of standard.
3. the external organ culture method of mammal embryo lung according to claim 1 is characterized in that: each interporal lacuna using the porous culture plate adds moisture equilibrium liquid, to slow down the substratum volatilization.
4. the external organ culture method of mammal embryo lung according to claim 1 is characterized in that: the adjustment carrying tablet is in the center of the bottom of culture hole after adding substratum among the step b, and the mammal embryo lung is positioned at the middle part of carrying tablet.
5. the external organ culture method of mammal embryo lung according to claim 1; It is characterized in that: the mode that the substratum described in the step c adopts half amount to increase progressively is changed liquid; Keep the half the in former culture hole of culture volume last time when promptly changing liquid at every turn, then will last time half of the culture volume fresh culture that by volume increases 4%-16% more than the mark add culture hole.
6. the external organ culture method of mammal embryo lung according to claim 1 is characterized in that: the described carrying tablet of step a is the transparent glass disk.
7. the external organ culture method of mammal embryo lung according to claim 1 is characterized in that: said moisture equilibrium liquid is aseptic phosphate buffered saline buffer or zero(ppm) water.
8. the external organ culture method of mammal embryo lung according to claim 1 is characterized in that: said embryo lung is the mice embryonic lung of 10～13 days isolating embryonic stages.
9. the external organ culture method of mammal embryo lung according to claim 1 is characterized in that: embryo lung is kept flat.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106967672A (en) * | 2017-03-24 | 2017-07-21 | 四川大学华西医院 | A kind of lung and cancerous lung tissue cultural method and with its build lung cancer in mice Animal models |
Citations (2)
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| US5344454A (en) * | 1991-07-24 | 1994-09-06 | Baxter International Inc. | Closed porous chambers for implanting tissue in a host |
| US5453278A (en) * | 1991-07-24 | 1995-09-26 | Baxter International Inc. | Laminated barriers for tissue implants |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5344454A (en) * | 1991-07-24 | 1994-09-06 | Baxter International Inc. | Closed porous chambers for implanting tissue in a host |
| US5453278A (en) * | 1991-07-24 | 1995-09-26 | Baxter International Inc. | Laminated barriers for tissue implants |
Non-Patent Citations (2)
| Title |
|---|
| O.A.TROWELL: "The culture of mature organs in a synthetic medium", 《EXPERIMENTAL CELL RESEARCH》, vol. 16, 31 December 1959 (1959-12-31), XP024790337, DOI: doi:10.1016/0014-4827(59)90201-0 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106967672A (en) * | 2017-03-24 | 2017-07-21 | 四川大学华西医院 | A kind of lung and cancerous lung tissue cultural method and with its build lung cancer in mice Animal models |
| CN106967672B (en) * | 2017-03-24 | 2021-01-26 | 四川大学华西医院 | Lung and lung cancer tissue culture method and method for constructing lung cancer mouse animal model by using same |
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