Name: Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching (Video) | JoVE Journal
Uploaded: 2010-06-30T17:00:00.000+00:00
Duration: 7 min 32 s
Description: Learn mouse embryonic lung culture for studying epithelial-mesenchymal interactions in developmental biology. This method enables observation of branching morphogenesis and treatment effects using liquid air interface culture.

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5 minutes

This work was supported by the Saban Research Institute Pre-doctoral Award (PMDM), and by NIH RO1 HL75773 (DW).

Organ culture is a very useful and versatile technique to study gene and protein expression. These ex vivo culture methods can be used as preliminary or in complement to in vivo studies.
1. Embryonic Lung Isolation
<ol>
 <li>Timed-pregnant mice are sacrificed on postcoitum day 11.5 or 12.5 (E11.5-12.5) by CO2 narcosis according to the NIH and OLAW guidelines. The animal is placed in a chamber and 100% CO2 is introduced. After the animal is unconscious the CO2 flow is increased. Clinical death of the animal must be ensured.</li>
 <li>All the following steps need to be completed under sterile conditions in a laminar flow hood. The uterus is removed from the pregnant females. To remove the uterus from the animal, the pregnant females are placed on their back and sprayed with 70% ethanol. An incision is made in the abdomen, and the skin is removed by pulling the skin upwards while holding the animal's hind legs. The peritoneal cavity is opened and the uterus is removed from the animal.</li>
 <li>The blood is rinsed off by placing the uterus in a 50 ml conical tube filled with cold Hanks Balanced Salt Solution (HBSS), and the tube is gently rocked for 2 minutes.</li>
 <li>The uterus is then placed in a Petri dish under the stereoscopic dissecting microscope, and the embryos released from the uterus by incising the uterine wall using spring scissors. The embryos are harvested using a perforated spoon, and placed on ice in a new Petri dish containing HBSS.</li>
 <li> Under the stereoscopic dissecting microscope, embryonic lungs are dissected one by one in a Petri dish containing cold HBSS (Figure 1).</li>
 <li>The isolated embryonic lung is placed on ice in a Petri dish containing HBSS using a sterile transfer pipette.</li>
</ol>
2. Embryonic Lung Culture
<ol>
 <li>500 microliters to 1 milliliter per well of D-MEM/F-12 supplemented with 50 units/ml of Penicillin-Streptomycin is added in a Nunclon Polystyrene dish.</li>
 <li>A Nuclepore Polycarbonate Track-Etch Membrane is placed on top of the media. When the Nuclepore Polycarbonate Track-Etch Membrane is placed on top of the media, make sure that the shiny side is against the media, and rough side is facing upwards.</li>
 <li>A dissected embryonic lung is placed on top of the Nuclepore Polycarbonate Track-Etch Membrane using a sterile transfer pipette.</li>
 <li>The embryonic lung position is adjusted using the forceps. Embryonic lung placed on the filter should have an intact trachea and be placed with their trachea having a straight position, thus allowing all lungs to have the same internal pressure.</li>
 <li>The Nunclon Polystyrene dish is transferred for the desired culture time in a cell incubator (Figure 2).</li>
</ol>
3. Materials
1. Embryonic Whole Lung Isolation
<ol>
 <li>Timed-pregnant (C57BL6 wild type or transgenic) mice to be sacrificed on postcoitum day 11.5 to 13.5 (E11.5-13.5).</li>
 <li>Hanks' Balanced Salt Solution (HBSS) (1X), liquid, without calcium chloride, magnesium chloride, or magnesium sulfate. (Invitrogen, Carlsbad, CA) supplemented with 50 units/ml of Penicillin-Streptomycin (Invitrogen, Carlsbad, CA). Store the HBSS at room temperature and at 4°C after opening. Aliquot and store the Penicillin-Streptomycin at -20°C.</li>
 <li>Stereoscopic dissecting microscope (Leica, Wetzlar, Germany).</li>
 <li>Dissection tools including Dumont forceps, Fine iris scissors (straight), Noyes spring scissors and Moria® spoon (perforated) (Fine Science Tools, Foster City, CA).</li>
</ol>
2. Embryonic Whole Lung Culture
<ol>
 <li>Dulbecco's Modified Eagle Medium:Nutrient Mix F-12 (D-MEM/F-12) (1X), liquid, 1:1 Contains L-glutamine, but no HEPES buffer. (Invitrogen, Carlsbad, CA) supplemented with 50 units/ml of Penicillin-Streptomycin (Invitrogen, Carlsbad, CA). Keep the DMEM/F-12 bottle away from the light (e.g. covered by aluminum foil) and store at 4°C. Aliquot and store the Penicillin-Streptomycin at -20°C. </li>
 <li>Nuclepore Polycarbonate Track-Etch Membrane, 8.0-m, 13mm (Whatman Nuclepore Track-Etch membrane (Whatman, Florham Park,
 NJ).</li>
 <li>Nunclon Polystyrene dish with lids, 4 wells (Rochester, NY).</li>
 <li>Disposable transfer pipettes, sterile (Fisher Scientific, Pittsburgh, PA).</li>
</ol>
<img src="/files/ftp_upload/2035/2035fig1.jpg" alt="Embryo development stages, E12.5, diagram: limb removal, organ identification, pharynx, trachea analysis." data-altupdatedat="1788235205000" data-alt="Figure 1"> Figure 1. Dissection of E12 embryonic lungs. (A) E12.5 whole embryo viewed from the right side. (B) Right forelimb has been removed from the embryo. (C) Forceps held by the left hand holding the embryo steady in the dish. Arrows indicate plan of dissection (D) Embryonic lung lies posterior to the heart (removed) and anterior to the spine. This figure shows the lungs after skin and heart removal. (E) Pharynx removal allows separation of the intact lung from the embryo. (F) Extraneous pharyngeal tissue and esophagus have been trimmed away. Dissected E12.5 embryonic lung with intact trachea and larynx is shown. (Cr) Cranial lobe, (Med) Medial lobe, (Ca) Caudal lobe, (Acc) Accessory lobe, (Le) Left lobe.
<img src="/files/ftp_upload/2035/2035fig2.jpg" alt="Lung organoid growth over 48h, control vs FGF9; microscopy images showing structure changes." data-altupdatedat="1788235205000" data-alt="Figure 2"> Figure 2. FGF9 induces expansion of the mesenchyme and the dilation of the epithelium in lung grown in vitro. (A-C) E12.5 lung is grown for 48 hours in absence of FGF9. Note the increase in branching. (D-F) E12.5 lung grown for 48 hours in presence of FGF9. Note the dilation of the epithelium and mesenchyme as early as after 24 hours of culture (E). After 48 hours (F), the effect on the epithelium is even more pronounced. Scale bar: A-F 400 μm 4.
The complete text protocol for this experimental approach is available in <a href="http://springerprotocols.com/Abstract/doi/10.1007/978-1-59745-019-5_5">Springer Protocols</a>.

<ol><li>Alescio, T., Cassini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induction+in+vitro+of+tracheal+buds+by+pulmonary+mesenchyme+grafted+on+tracheal+epithelium%5BTitle%5D)+AND+%22J+Exp+Zool%22%5BJournal%5D)">Induction in vitro of tracheal buds by pulmonary mesenchyme grafted on tracheal epithelium.</a> J Exp Zool. 150, 83-94 (1962).</li>
<li>Warburton, D., Schwarz, M., Tefft, D., Flores-Delgado, G., Anderson, K. D., Cardoso, W. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+molecular+basis+of+lung+morphogenesis%5BTitle%5D)+AND+%22Mech+Dev%22%5BJournal%5D)">The molecular basis of lung morphogenesis.</a> Mech Dev. 92, 55-81 (2000).</li>
<li>Spooner, B. S., Hardman, P., Paulsen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gravity+in+mammalian+organ+development%3A+differentiation+of+cultured+lung+and+pancreas+rudiments+during+spaceflight%5BTitle%5D)+AND+%22J+Exp+Zool%22%5BJournal%5D)">Gravity in mammalian organ development: differentiation of cultured lung and pancreas rudiments during spaceflight.</a> J Exp Zool. 269, 212-222 (1994).</li>
<li>Moral, del, M, P., De Langhe, S. P., Sala, F. G., Veltmaat, J. M., Tefft, D., Wang, K., Warburton, D., Bellusci, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+role+of+FGF9+on+epithelium+and+mesenchyme+in+mouse+embryonic+lung%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">Differential role of FGF9 on epithelium and mesenchyme in mouse embryonic lung.</a> Dev Biol. 293, 77-89 (2006).</li>
<li>Moral, D. el, M, P., Sala, F. G., Tefft, D., Shi, W., Keshet, E., Bellusci, S., Warburton, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((VEGF-A+signaling+through+Flk-1+is+a+critical+facilitator+of+early+embryonic+lung+epithelial+to+endothelial+crosstalk+and+branching+morphogenesis%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">VEGF-A signaling through Flk-1 is a critical facilitator of early embryonic lung epithelial to endothelial crosstalk and branching morphogenesis.</a> Dev Biol. 290, 177-188 (2006).</li>
<li>Jaskoll, T. F., Don-Wheeler, G., Johnson, R., Slavkin, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Embryonic+mouse+lung+morphogenesis+and+type+II+cytodifferentiation+in+serumless%2C+chemically+defined+medium+using+prolonged+in+vitro+cultures%5BTitle%5D)+AND+%22Cell+Differ%22%5BJournal%5D)">Embryonic mouse lung morphogenesis and type II cytodifferentiation in serumless, chemically defined medium using prolonged in vitro cultures.</a> Cell Differ. 24, 105-117 (1988).</li>
<li>Seth, R., Shum, L., Wu, F., Wuenschell, C., Hall, F. L., Slavkin, H. C., Warburton, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+epidermal+growth+factor+expression+in+early+mouse+embryo+lung+branching+morphogenesis+in+culture%3A+antisense+oligodeoxynucleotide+inhibitory+strategy%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">Role of epidermal growth factor expression in early mouse embryo lung branching morphogenesis in culture: antisense oligodeoxynucleotide inhibitory strategy.</a> Dev Biol. 158, 555-559 (1993).</li>
</ol>

No conflicts of interest declared.

mouse embryonic lung culture, a system to evaluate the molecular mechanisms of branching

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.

Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of ...

In this video, a method for the dissection and culture of mouse embryonic lungs is demonstrated. The procedure begins with the harvesting of embryos at E 11.5 or E 12.5. Next, the embryos are opened and the lungs are extracted.The lungs are then plated in a liquid air interface and incubated following culture. Changes in the growth and branching of mesen chy and epithelium of the embryonic lung in response to specific treatments can be observed using a stereo microscope. Hello, I'm David War Burton.I direct the developmental biology and regenerative medicine program at the Ban Research Institute Children's Hospital, Los Angeles. And I'm Jan Carro, a senior postdoc in Dr.War Burton Lab. Today we are going to show you a technique for isolating embryonic mouse lung and placing them in organ culture.This technique has evolved from the original description by Tina Jaco Halkin and Pablo bring us at the USC School of Dentistry about 20 years ago. We use this procedure in our lab to study lamb branching morphy. So let's get started.On Postum Day 11.5 or 12.5, place a carbon dioxide ssis sacrificed timed pregnant mouse under a laminar flow hood on its back. Spray with 70%ethanol. Make an incision in the abdomen.Then while holding the animal's hind legs, remove the skin by pulling upwards. Open the peritoneal cavity and remove the uterus. Place the uterus in a 50 milliliter conical tube filled with cold hanks balanced salt solution or HBSS and gently rock for two minutes to rinse off the blood.Next, to transfer the uterus to a Petri dish under a stereoscopic dissecting microscope. Using spring scissors, cut the uterine wall to release the embryos. Then using a perforated spoon, harvest the embryos and place them in a Petri dish containing HBSS and place on ice.Continue working under the stereoscopic dissecting microscope and begin dissecting embryonic lungs. Begin by placing the embryo on its left side. Using forceps, remove the right forelimb holding forceps in the left hand.Cut the embryo starting from the limb opening and remove the skin. This step will allow you to see internal organs and to localize the heart and lungs for the following step. Remove the heart.The embryonic lungs are located posterior to the heart and anterior to the spine. Next, separate the intact lung from the embryo. Then trim away extraneous tissue and the esophagus.Once the lungs have been removed, use a sterile transfer pipette to transfer them to a Petri dish containing cold HBSS on ice. To each well of a no clone delta polystyrene dish at 0.5 to one milliliter of D-M-E-M-F 12 supplemented with 50 units per milliliter of penicillin streptomycin. Next place a nucleo polycarbonate track etch membrane on top of the media, shiny side down and rough side up.Using a sterile transfer pipette place a dissected embryonic lung on top of the nucleo polycarbonate track etch membrane. Using the stereo microscope, adjust the embryonic lung position. The embryonic lung should have an intact trachea placed in a straight position as shown here.This ensures that all of the lungs have the same internal pressure, transfer the dish to a cell incubator and culture for the desired time. Here, E 12.5 lungs were grown in the presence or absence of FGF nine. FGF nine is a member of the fibroblast growth factor family that induces expansion of the mechy and the dilation of the epithelium in lungs grown in vitro after 48 hours.In the absence of FGF nine, increased branching is seen. These images are photographs of the embryonic lung taken under a stereo microscope. In contrast, lungs grown 48 hours in the presence of FGF nine have dilated epithelium and mechy.After 24 hours of culture. After 48 hours, the effect on the epithelium is even more pronounced. So we have just shown you how to prepare organ cultures from embryonic mouse lung.When doing this procedure is important to remember to remove the the lung from the embryo as soon as possible to reduce the tissue deterioration. So thanks for watching. Hopefully you'll find this a useful technique.Good luck with your experiments.

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Learn mouse embryonic lung culture for studying epithelial-mesenchymal interactions in developmental biology. This method enables observation of branching morphogenesis and treatment effects using liquid air interface culture.

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

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