CN102391984A - Embryo lung in vitro culture method - Google Patents

Abstract

The invention belongs to the field of biotechnology, and relates to an in vitro organ culture method of mammalian embryonic lung, in particular to an in vitro organ culture method for mouse embryonic lung. The technical problem to be solved by the present invention is mainly the defect that the existing culture method requires special materials and occupies a large space. The technical solution of the present invention is an in vitro organ culture method for embryonic lungs, comprising the following steps: a. Putting a carrier sheet with a size adapted to it in the culture hole of the multi-well culture plate, and constructing a culture space at the bottom of the culture hole; b. The obtained mammalian embryonic lungs may be placed on a carrier sheet, and culture medium is added to make the lungs at the junction of the medium and air; c, cultured in a 37°C incubator, and then the medium is changed every 1 to 2 days. Using the method of the invention can obtain a large number of in vitro cultured lung organs that develop stably and are convenient for subsequent operations at low cost, and can also meet the needs of large-scale drug screening.

Description

In vitro culture method of embryonic lung

technical field

The invention belongs to the field of biotechnology, and in particular relates to an in vitro organ culture method for mammalian embryonic lungs, in particular to an isolated organ culture method for mouse embryonic lungs.

Background technique

The study of mammalian organ development enables the understanding of several fundamental cell biological processes in the context of intact organs. This is especially important in the age of genetics, since gene deletions or mutations in mammals such as mice etc. can be directly linked to congenital malformations in humans.

In vitro organ culture is an advanced form of tissue culture. It can reproduce the development process of organs and simulate the functional state of organs in different states and conditions. It is an important technology for understanding organ development process, researching disease pathogenic mechanism and drug screening. means. This is particularly useful for organoid lungs cultured via a branching morphogenetic mechanism. The development of the mouse embryonic lung begins with the protrusion of the laryngotracheal groove in the foregut endoderm at embryonic day 9.5, and then the formation of the sprout base gradually forms the bronchial branch. At this time, each bud base contains three layers, the epidermis, the surrounding mesenchyme and mesothelial layer, and finally the lungs of the respiratory system. The branching, stratification and organogenesis of the lung require the regulation of cytokines and signaling pathways such as epidermal growth factor (EGF) and fibroblast growth factor (FGF). In order to determine which factors have an influence on these processes, the in vitro culture experimental system of early embryonic lung came into being.

The currently more common in vitro culture system of embryonic lungs is based on Trowell's 1959 The culture of mature organs in a synthetic medium. Exptl. CeZZ Res. 16: 118-147, 1959. Many people improved it later, but its core In order to utilize a large-diameter petri dish (35mm, 60mm), add an appropriate amount of medium, and float a polycarbonate filter membrane with a pore size of about 5um on the medium. Then mouse embryonic lungs were placed on the filter membrane and cultured at the air-liquid junction (Fig. 1, 2, 3, 4). However, the use of the above method requires (1) the use of special materials such as polycarbonate filter membranes, (2) the waste of a large amount of culture dish space, (3) the limited contact area between the embryonic lung and the medium is not conducive to further effective experimental intervention, ( 4) And it is not conducive to the application of large-scale embryonic lung culture.

Contents of the invention

The purpose of the invention is to solve the problems that conventional culture methods need to use special materials such as polycarbonate filter membranes and waste a lot of culture dish space.

The solution of this program is to provide an in vitro organ culture method of mammalian embryonic lung, which is characterized in that it comprises the following steps:

a. Put a carrier sheet with a suitable size in the culture well of the multi-well culture plate, and build a culture space at the bottom of the culture well;

b. placing the obtained mammalian embryonic lung on a carrier sheet, adding culture medium, so that the embryonic lung is at the liquid-gas interface between the culture medium and air;

c. Cultivate in an incubator at 36°C to 38°C, and then change the medium every 1 to 2 days.

Wherein, the multi-well culture plate described in step a of the above method is a standard 24-well culture plate (arranged in 4×6 wells, the diameter of the wells is 15.6 mm, and the area of the bottom of the wells is 1.9 m ² ).

Wherein, the multi-hole culture plate described in step a of the above method is only a container for the liquid medium, and does not directly contact with the embryonic lung, and a domestically sterilized standard 24-well culture plate can be used.

Wherein, the carrier sheet described in step a of the above method is a transparent glass disc. In direct contact with cultured embryonic lungs, corresponding cell biology treatments should be performed. The size of the carrying sheet is slightly smaller than the bottom area of the culture well, which is 50-80% of the size of the culture well.

Wherein, in step a of the above method, a humidity balance liquid is added between the holes of the multi-well culture plate to slow down the volatilization of the culture medium.

Wherein, the humidity balance liquid in the above method is sterile phosphate buffer saline or distilled water.

Wherein, the embryonic lung described in step b of the above method is the isolated mouse lung primordia of embryonic days 10-13.

Wherein, the way of placing the lungs in step b of the above method is: laying the embryonic lungs flat. The embryonic lungs were placed at the liquid-air junction of the medium.

Wherein, after the medium is added in step b of the above method, the carrier sheet is adjusted at the center of the bottom of the culture well, and the mammalian embryonic lung is located at the middle of the carrier sheet.

Wherein, the culture medium described in step c of the above-mentioned method is changed in a half-volume-increasing manner, that is, half of the previous culture medium volume is kept in the original culture well when the medium is changed each time, and then half of the previous culture medium volume is replaced by half of the previous culture medium volume. Add 4%-16% more fresh medium to the culture wells by volume fraction. It is preferred to increase the medium volume by 8%-13%, optimally by 10%-12%.

The beneficial effect of the method of the present invention is that it overcomes the disadvantage of large space occupation of all the current methods, greatly improves the space utilization rate, reduces the cost, and is convenient for large-scale cultivation. The efficiency of experimental interventions is increased due to the greater exposure of the lungs to the culture medium. Small-volume culture reduces the amount of medium used, only about 180ul of medium is needed for culture. It overcomes the prejudice that the existing technology does not require expensive imported materials, imported filter membranes, and takes up a large cultivation space and resources to achieve good results, and the cost is greatly reduced. When doing experimental intervention, it is also possible to reduce the amount of intervention reagents to achieve the desired concentration or effect. The assembly and operation are simple, just put the treated cover glass into the culture well, place the embryonic lung, and add the medium for culture, which is simple and easy. There is a better culture shape of the lungs. Since there is no cover of the supporting membrane, it is convenient to observe the tracheal branches of the lungs. For example, a general cell inverted microscope can be used to observe the culture of the lungs in vitro. The added cover glass also facilitates subsequent operations, such as in situ hybridization, immunofluorescence and other operations. The method of the invention cultivates the embryonic lung in vitro for more than 10 days, and the bronchial smooth muscle of the lung can contract rhythmically, and has good application prospect.

Description of drawings

Fig. 1. System schematic diagram of the existing embryonic lung culture method. 1. 60mm culture dish; 2. Embryonic lung; 3. Polycarbonate membrane; 4. Culture medium.

Fig. 2. System schematic diagram of the existing embryonic lung culture method. 1. 60mm culture dish; 2. Embryonic lung; 3. Polycarbonate membrane; 4. Culture medium.

Fig. 3 is a schematic diagram of the system of the embryonic lung culture method of the present invention. 1. Culture wells on 24-well culture plate; 2. Embryo lung; 3. Culture medium; 4. Carrier sheet.

Fig. 4 is a schematic diagram of the system of the embryonic lung culture method of the present invention. 1. Culture wells on 24-well culture plate; 2. Embryo lung; 3. Culture medium; 4. Carrier sheet.

Figure 5 shows the growth of mouse embryos at the embryonic stage of 12.5 days after being cultured for ten days.

Figure 6 is a video screenshot showing that on the fifth day of embryonic lung culture, the bronchial smooth muscle of the lungs showed rhythmic contraction after that, and the parts indicated by the arrows were the parts with a fairly obvious range of motion, which were respectively located in the large bronchi and two of the lungs. Small tracheal branches of the lung lobes.

Figure 7. Protocol for changing media volumes.

Figure 8. A picture of a standard 24-well cell culture plate, showing the gaps between the wells for humidifying the equilibrium solution. A gap is formed between every 4 adjacent culture wells, and the standard 24-well cell culture plate has 15 gaps where humidity balance solution can be added. In the figure, 1 is the gap between the culture wells, and 2 is the culture well.

Figure 9. A photograph of a standard 24-well cell culture plate to show the gaps between the wells for humidifying the equilibrium solution. A gap is formed between every 4 adjacent culture wells, and the standard 24-well cell culture plate has 15 gaps where humidity balance solution can be added. In the figure, 1 is the gap between the culture wells, and 2 is the culture well.

Detailed ways:

The present invention will be described in detail below through specific embodiments in conjunction with the accompanying drawings.

The invention provides a method for in vitro organ culture of mammalian embryonic lung, comprising the following steps:

a. Put a carrier sheet with a suitable size in the culture well of the multi-well culture plate, and build a culture space at the bottom of the culture well;

b. placing the obtained mammalian embryonic lung on a carrier sheet, adding culture medium, so that the embryonic lung is at the liquid-gas interface between the culture medium and air;

c. Cultivate in an incubator at 36°C to 38°C, and then change the medium every 1 to 2 days.

Wherein, the multi-well culture plate described in step a of the above method is a standard 24-well culture plate (arranged in 4×6 wells, the diameter of the wells is 15.6 mm, and the area of the bottom of the wells is 1.9 m ² ).

Wherein, the multi-hole culture plate described in step a of the above method is only a container for the liquid medium, and does not directly contact with the embryonic lung, and a domestically sterilized standard 24-well culture plate can be used.

Wherein, the carrier sheet described in step a of the above method is a transparent glass disc, which is in direct contact with the cultured embryonic lung and must be subjected to corresponding general cell biology treatment. The size of the carrier sheet should be smaller than the size of the well, 50-80% of the size of the culture well, and the shape should match. It is best to use a round transparent glass disk like the culture well. The carrier sheet should generally be attached to the bottom.

Wherein, in the above method, a humidity balance solution can be added between the holes of the multiwell culture plate used to slow down the volatilization of the culture medium.

Wherein, the humidity balance liquid in the above method is sterile phosphate buffer saline or distilled water.

Wherein, the embryonic lung described in step b of the above method is the mouse embryonic lung isolated from the embryonic period of 10-13 days.

Wherein, the way of placing the lungs in step b of the above method is: laying the embryonic lungs flat. The embryonic lungs were placed at the liquid-air interface of the culture medium.

Wherein, after the medium is added in step b of the above method, the carrier sheet is adjusted at the center of the bottom of the culture well, and the mammalian embryonic lung is located at the middle of the carrier sheet.

Wherein, the culture medium described in step c of the above-mentioned method is changed in a half-volume-increasing manner, that is, half of the previous culture medium volume is kept in the original culture well when the medium is changed each time, and then half of the previous culture medium volume is replaced by half of the previous culture medium volume. Add 4%-16% more fresh medium to the culture wells by volume fraction.

Various reagents and materials used in the method of the present invention should be sterile according to common knowledge in the field, and the operation should also be carried out under sterile conditions.

The carrier sheet used in the method of the present invention can be a thin sheet suitable for tissue cell culture of various materials, and the most commonly used and optimal one is a transparent glass disc. The carrier sheet should be fully cleaned and sterilized before use. If necessary, it can be pretreated with a suitable matrix, such as polylysine, gelatin or collagen.

The multi-hole culture plate used in the method of the present invention can be a conventional common 24-well plate that can be applied to cell tissue culture, and the holes are preferably flat-bottomed round holes with a bottom area of 1 to 3 square centimeters. The most common is the standard 24-well flat-bottom cell culture plate, which can be made of glass or plastic. The standard specification is 4X 6 hole arrangement cloth, the hole diameter is 15.6mm, and the hole bottom area is 1.9m ² .

The humidity balance liquid used in the method of the present invention must be sterile and non-toxic. It is best to use sterile phosphate buffered saline or distilled water, and the amount to be used depends on the multi-well culture plate used. There is a gap (see Fig. 8, Fig. 9) between the wells of the multi-well culture plate, which can be used to add humidity balance liquid in the present invention, and the volume of the humidity balance liquid used in the gap between each hole of a general standard 24-well flat-bottomed cell culture plate should be greater than 200ul , preferably 500ul.

The source of the embryonic lung used in the method of the present invention is the embryo of a mouse commonly used in experiments. The experimental mouse embryonic lung can be the lung primordium of embryonic days 10-13 (lung primordium is a name for embryonic lung in the early development stage in this field, and generally includes the trachea and lung lobes branched from the esophagus). And preferably only one embryonic mouse lung is cultured on each carrier sheet. It is best to lay the embryonic lung flat, that is, place it horizontally in the center of the carrier sheet according to the cross section so that the lung primordium can develop into a good shape. After the medium is added, the embryonic lungs are placed at the liquid-air interface between the medium and the air. This description in this field means that the medium cannot completely submerge the embryonic lungs, but it cannot be directly exposed to the air, but To make it form a liquid film of a culture medium on the upper surface, this is called the liquid-gas interface between the culture medium and air.

When obtaining the embryonic lung, it is necessary to ensure the integrity of the embryonic lung during the operation (do not destroy the tracheal branch of the embryonic lung to avoid abnormal lung development).

The culture medium used in the method of the present invention can be various cell culture mediums commonly used in the art, such as dulbecco's modifi edeagle's medium (DMEM, a general culture medium, such as the product of the merchant's Invitrogen product number 11965, or can also refer to what is disclosed on the Internet: http: //baike.baidu.com/view/3501452.htm DMEM (H) cell culture medium (powder type) composition preparation) or Eagle's minimal essential medium (EMEM, general medium, known formula. Then in the selected culture Add FBS (fetal bovine serum) or F12 nutrient factor (product of Invitrogen Company, product name: nutrient factor mixture, item number: 21700) with a volume fraction of 5-20% in the base, and generally can also add antibiotics at a final concentration of 0.1mg/ml penicillin and streptomycin (penicillin and streptomycin) to prevent contamination.

The culture conditions in the method of the present invention are general cell culture conditions. The general temperature is 36°C to 38°C; it is best to cultivate at 37°C in an environment with a volume fraction of 5% carbon dioxide.

The culture medium used in the method of the present invention can remove the old culture medium and add an equal volume of new culture medium to continue culturing every 24 to 48 hours. The preferred method of changing the liquid that can also be adopted is to change the liquid in a half-volume incremental manner, that is, keep half of the previous culture medium volume in the original culture well when changing the liquid each time, and then return half of the previous culture medium volume by volume. Fractions increased by 4%-16% fresh medium was added to culture wells. It is preferred to increase the medium volume by 8%-13%, optimally by 11%. If a standard 24-well flat-bottomed cell culture plate is used, the medium volume can be 150-200ul per well, and the optimal volume should be 180ul per well. When changing the medium, add 5-15ul fresh medium that is half the volume of the previous medium into the culture well. Preferably 8-12ul, most preferably 10ul.

Example 1: Using the method of the present invention to culture Balb/c mouse embryonic day 11.5 lung.

1. Prepare the carrier: use a glass dome with a diameter of 12mm as the carrier, and buy it from Haimen Huakai Experimental Glass Instrument Co., Ltd. First, prepare a 1% hydrochloric acid solution with distilled water, shake and clean the dome at room temperature for one hour. After cleaning with distilled water, put it into a 70% alcohol solution by volume, shake and clean overnight. Use a 10cm glass plate to hold the cleaned domes, drain the liquid, put it in a high-temperature steam sterilizer, and sterilize at 121°C for 40 minutes. After drying, put it on the ultra-clean workbench for later use.

2. Assemble the culture device: use sterile tweezers to pick up the processed dome and place it in the culture well of a sterile 24-well culture plate. The sterile 24-well culture plate was 3524 tissue culture plate purchased from Costar (Corning, USA).

3. Obtain mouse lungs at embryonic day 11.5: Pregnant Balb/c female mice were obtained from the SPF level (no special pathogenic bacteria) animal room of the Genetic Engineering Mouse Center of West China Hospital of Sichuan University. The day when the vaginal suppository was detected was recorded as E0.5 On day E11.5, the embryonic development time was calculated, and the pregnant mice were taken out from the animal breeding room on day E11.5. After the pregnant mice were killed by necking, the pregnant mice and dissection instruments were disinfected with alcohol. After taking out the mouse embryos, dissect them in pre-cooled dissecting solution (1X PBS, HBSS or DMEM) on ice, take out the embryonic lungs under a stereoscope, and transfer them to the culture medium (DMEM+10% FBS) 1.5 with a 1mL suction tip. ml in EP tubes.

4. In the ultra-clean workbench, use a 1mL pipette to transfer the embryonic lungs to the culture wells, one for each well. Use a pipette to suck away the residual medium in the culture well and add 180ul medium. Use the high temperature sterilized tweezers with an alcohol lamp, adjust the embryonic lung to the middle of the cover piece after cooling down, and adjust the position of the cover piece so that the embryonic lung is in the middle of the culture hole. The construction of the culture system is shown in Figure 3 and Figure 4 for schematic diagrams.

5. Carefully place the seeded embryonic lungs in a cell culture incubator at 37°C and 5% CO ₂ for 24 hours.

6. Use a pipette to keep 90ul of the original medium in the culture well, and discard the remaining medium.

7. Add 90+10ul of fresh medium to the culture well, mix well and culture in the cell culture incubator.

8. After culturing for 24 hours, perform the same step 6 and 7 to perform a half-volume incremental change of medium: keep 95ul of the original medium + 105ul of the new medium.

9. Change the liquid every 24 hours. The replacement medium volume scheme is shown in Figure 7.

10. The effect of culturing for 10 days (see accompanying drawing 5).

Figure 5 shows the growth of mouse embryos at day 12.5 after being cultured for ten days. The time for culturing the embryonic lung can be more than 10 days, and the pulmonary trachea branches are fully branched and expanded, and a good shape is obtained.

Figure 6 is a video screenshot showing that on the fifth day of embryonic lung culture, the bronchial smooth muscle of the lungs showed rhythmic contraction after that, and the parts indicated by the arrows were the parts with a fairly obvious range of motion, which were respectively located in the large bronchi and two of the lungs. Small tracheal branches of the lung lobes. It shows that the cultured kidney is close to the situation in the body, and has certain advanced physiological functions.

After many tests, using the above method to use E12.5-day embryonic lungs for in vitro culture is almost 100% successful. Using the method of 1 embryonic lung per well, 24 wells can be cultured in all wells of a 24-well plate. The best is Use the wells in the inner circle of 8 for cultivation. 24 embryonic lungs were cultured in a 24-well plate (13cmX8.5cmX2.5cm). That is, there is one lung in a space of about 11 cubic centimeters, and the minimum in the existing technology is generally 3.5cmX3. Strictly controlled operations in the same environment lead to better growth consistency and facilitate large-scale drug screening.

Claims (9)

1. the in vitro organ culture method of mammalian embryo lung, it is characterized in that comprising the following steps: a. Put a carrier sheet with a suitable size in the culture well of the multi-well culture plate, and build a culture space at the bottom of the culture well; b. placing the obtained mammalian embryonic lung on a carrier sheet, adding culture medium, so that the embryonic lung is at the liquid-gas interface between the culture medium and air; c. Cultivate in an incubator at 36°C to 38°C, and then change the medium every 1 to 2 days. 2. The in vitro organ culture method of mammalian embryonic lung according to claim 1, characterized in that: the multi-well culture plate described in step a is a standard 24-well culture plate. 3. The in vitro organ culture method of mammalian embryonic lung according to claim 1, characterized in that: a humidity balance solution is added between each hole of the multi-well culture plate to slow down the volatilization of the culture medium. 4. The in vitro organ culture method of mammalian embryonic lung according to claim 1, characterized in that: after adding medium in step b, adjust the carrier sheet at the center of the bottom of the culture hole, and the mammalian embryonic lung is located in the middle of the carrier sheet . 5. The in vitro organ culture method of mammalian embryonic lung according to claim 1, characterized in that: the medium described in step c is changed in a half-amount increasing manner, that is, the last culture medium is kept when changing the liquid each time Half of the volume is in the original culture well, and then a fresh medium that is 4%-16% more than the half of the previous culture medium volume is added to the culture well. 6. The method for in vitro organ culture of mammalian embryonic lung according to claim 1, characterized in that: the carrier sheet described in step a is a transparent glass disc. 7. The in vitro organ culture method of mammalian embryonic lung according to claim 1, characterized in that: the humidity balance liquid is sterile phosphate buffer saline or distilled water. 8 . The method for in vitro organ culture of mammalian embryonic lung according to claim 1 , characterized in that: the embryonic lung is isolated mouse embryonic lung of 10-13 days embryonic stage. 9. The method for in vitro organ culture of mammalian embryonic lung according to claim 1, characterized in that: the embryonic lung is laid flat.

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