The authors were supported by: a CancerFree KIDS Research Grant, a March of Dimes Basil O&#8217;Connor Starter Scholar Award (#5-FY14-32), a Cincinnati Children&#8217;s Hospital Medical Center (CCHMC) Trustee Grant Award; a CCHMC Research Innovation and Pilot Funding Award; and CCHMC developmental funds. Authors also acknowledge the Capel laboratory for the initial optimization of this technique.

All mice used in these studies were CD-1 mice obtained from Charles River Laboratories. Previous culture experiments have also been performed on other strains, such as C57BL/6J (data not shown), but any strain can be used. Pregnant adult females were approximately 2-3 months old and were euthanized via CO2 inhalation followed by cervical dislocation and bilateral thoracotomy prior to embryo removal. Mice were housed in accordance with NIH guidelines, and experimental protocols were approved by the Institutional Animal Care and Use Committee of Cincinnati Children&#8217;s Hospital Medical Center.
1. Preparation of Instruments, Culture Media, and Dishes
<ol>
	<li>Prepare a 70% ethanol solution. Autoclave deionized water (for making humidified chambers and for making/diluting solutions). Sterilize dissection tools (forceps, scissors, and 27 G needle syringes) by spraying down with 70% ethanol.</li>
	<li>Prepare 10X phosphate-buffered saline (PBS) containing calcium and magnesium (80 g NaCl, 2 g KCl, 14.4 g Na2HPO4, 2.4 g KH2PO4, 6.75 ml&#160;of 1 M CaCl2, 3.75 ml&#160;of 1 M MgCl2). Stir to dissolve in 1 L sterile autoclaved water and then filter with filter paper. Dilute 10-fold with water to make 1X PBS.</li>
	<li>Heat inactivate fetal bovine serum (FBS) at 55 &#176;C for 30 min and filter sterilize with a 0.22 &#181;m syringe filter.</li>
	<li>Prepare 38 ml&#160;1X complete culture medium (called complete DMEM, cDMEM), consisting of Dulbecco&#8217;s Modified Eagle Medium (DMEM), 10% filtered heat-inactivated FBS and 1/100 dilution of penicillin-streptomycin stock. This usually should be used fresh, but can be stored at 4 &#176;C for 1 month.</li>
	<li>Reconstitute the VEGFR Tyrosine Kinase Inhibitor II (TKI II) in DMSO for a stock concentration of 5 mg/ml, and dilute stock with cDMEM to make a working solution. The final concentration for this study is 1.875 &#181;g/ml&#160;in cDMEM. According to the company&#8217;s recommendations, stock solutions are stable for up to 6 months at -20 &#176;C. As for working solutions, make fresh and use on the same day. 
	Note: The concentration of this drug in the working solution should be two-fold higher than the desired final concentration (since it will be diluted two-fold in the droplet). At the same time, prepare the same volume of cDMEM containing the same volume of DMSO for the &#8220;control&#8221; treatment.</li>
	<li>Prepare the tail digestion reagents (50 mM NaOH and 1 M Tris-HCl [pH 8.0]) separately in distilled water.</li>
	<li>Make a 25 &#956;M stock of the XY PCR primers (XY Forward 5&#39;-TGA AGC TTT TGG CTT TGA G-3&#39; and XY Reverse 5&#39;-CCG CTG CCA AAT TCT TTG G-3&#39;) together in nuclease-free water.</li>
	<li>Prepare 50X TAE buffer (242 g Trizma Base, 57.1 ml&#160;Glacial Acetic Acid, 100 ml&#160;0.5 M EDTA, pH 8.5, and add water to 1 L final volume). Prepare 1X TAE Running buffer (20 ml&#160;50X TAE diluted with water to 1 L total volume).</li>
	<li>To create a 2.5% agarose solution (0.625 g agarose, 0.5 ml&#160;50X TAE Buffer, 24.5 ml&#160;water), heat agarose solution in microwave until boiling, then let cool until about 55-65 &#176;C (able to touch). Once cool, add 2 &#181;l&#160;of 1% ethidium bromide solution, and pour gel. 
	CAUTION! Ethidium bromide is a mutagen and teratogen; please use nitrile gloves and proper safety protocol when handling. Alternatively, other potentially less toxic gel resolving agents or dyes can be used.</li>
	<li>Prepare Blocking Solution (PBS with 0.1% Triton X-100, 10% fetal bovine serum [FBS], and 3% bovine serum albumin [BSA]) for immunofluorescence.</li>
	<li>Prepare Washing Solution (PBS with 0.1% Triton X-100, 1% FBS, and 3% BSA) for immunofluorescence.</li>
	<li>Prepare PBTx (PBS with 0.1% Triton X-100) for immunofluorescence.</li>
	<li>Prepare the incubation chambers for culture. Fill large (100 mm diameter) petri dishes with 35 ml&#160;autoclaved water. Place near where dissection and cultures will be performed.</li>
</ol>
2. Isolation of Fetal Testes from Mus musculus
<ol>
	<li>Check the female mouse for vaginal plugs each morning. Noon on the day at which the vaginal plug is detected is considered E0.5. Euthanize the pregnant female mouse at E11.5, in a manner consistent with approved animal protocols.</li>
	<li>Spray down mouse abdomen with 70% ethanol.</li>
	<li>Open the belly skin and peritoneum with a V-shaped incision using fine scissors and pull back flap of tissue to expose interior organs.</li>
	<li>Locate the ovaries at both ends of the uterine horn. Using scissors, cut at the ovary and the connective tissue to separate the uterus containing the embryos from the mother&#8217;s body.</li>
	<li>Open the uterine wall by gently cutting the side opposite the placentae, exposing the yolk sacs. Be careful not to puncture yolk sac to avoid damaging or losing embryos.</li>
	<li>Cut near the placentae to remove the embryo-containing yolk sacs and place them into a dish containing PBS with calcium and magnesium. The presence of calcium and magnesium ions will help support cell adhesion molecules to maintain tissue architecture and prevent the tissue from sticking to the dissection tools.</li>
	<li>With forceps, remove the yolk sac and amnion from the embryo by carefully puncturing the yolk sac to create a hole in which the embryo can slide out. Once the embryo is outside of the yolk sac, cut the umbilical cord.</li>
	<li>From this point forward, use a microscope located in a sterile tissue culture hood. Remove the head of embryo and discard by pinching with forceps on either side of neck.</li>
	<li>Remove the tail and place into new microcentrifuge tube for XY PCR analysis to determine sex of embryo. 
	Note: While it is optimal to culture gonads as soon as possible after harvest, it is also possible to remove tail DNA and perform XX/XY genotyping prior to culture, so that the sex of each embryo is known and only the desired sex needs to be cultured. While the genotyping is being done, embryos can be kept separated (so that they can be tracked) at 4 &#176;C or on ice until ready for culture. One caveat is that this period outside in utero or culture conditions may potentially affect viability for culture or subsequent development during culture.</li>
	<li>Secure the embryo in a supine position against the bottom of the culture dish by pinning the armpits of embryo with one pair of forceps (usually the forceps held in the weak hand). Maintain these forceps in place to hold the embryo still during the entire dissection process.</li>
	<li>With other pair of forceps, remove skin covering abdomen and gently remove liver, intestines, and other organs to expose back body wall.</li>
	<li>As the gonads will be located on the back body wall on either side of the dorsal aorta, a large blood vessel running along the midline of the body, scoop underneath the urogenital ridge with closed forceps and remove the urogenital ridge by opening the forceps and lifting up. As the gonads will be loosely attached to the wall, be careful not to pull up too fast without removing these connections or the gonads may stretch, causing damage to the organs.</li>
	<li>Move the urogenital ridge into cDMEM in a dish to acclimate tissue to media.</li>
	<li>Separate the gonad-mesonephros complex from the rest of the urogenital ridge using 27 G needles. Use one needle to cut by pressing down, and use the other to guide the tissue correctly to allow optimal separation. Avoid using a sawing motion against the dish bottom, as sharp needles will create shards of plastic that can stick to the tissue. Make sure to keep the mesonephros completely attached to the gonad.</li>
</ol>
3. Culturing of Gonad with a Small-molecule Inhibitor
<ol>
	<li>Set two 20&#160;&#181;l&#160;pipettes to 15 &#181;l&#160;each. Cut about 1-2 mm off one of the barrier pipette tips using a clean, sterilized razor blade for transfer of the gonads and addition of control cDMEM, while the other pipette will be used solely for drug-treated cDMEM.</li>
	<li>Line up gonads with their long axis parallel to the pipette tip so they can easily be pipetted using the cut-off tip. Be careful of gonads sticking to the inside of the pipette tip.</li>
	<li>Label one side as the control droplet and the other as the drug-containing droplet. Only two droplets can fit on a 35 mm dish lid. Make sure that labels on lids match the labels on tail-tissue-containing microcentrifuge tubes.</li>
	<li>Pipette 15 &#181;l&#160;of cDMEM containing a single gonad into a droplet in the lid of a small (35 mm) culture dish (use only the lids, as the bottoms are too tall to fit within a humidified petri dish chamber). Place two separate gonad-containing droplets on either side of the dish, well-separated from one another. Check under the microscope to ensure that the gonads have been transferred to the droplets.</li>
	<li>To the droplet designated as the control, add an additional 15 &#181;l&#160;of cDMEM containing DMSO (made in step 1.5) to make a droplet with a 30 &#181;l&#160;total volume. Use only the &#8220;control&#8221; pipette that will not contact the drug.</li>
	<li>To the other droplet (drug-treated sample), add 15 &#181;l&#160;of TKI-II-containing cDMEM to make a droplet with a 30 &#181;l&#160;total volume. Use only the pipette designated for drug-treated samples.</li>
	<li>Using the pipette, spread out the droplets in an expanding circular pattern until they are about 15-18 mm in diameter and the gonad is located roughly in the middle. Orient the gonad so that it lies on its side and the gonad and mesonephros are easily distinguishable. Orient the lung so that the two lobes lay flat.
	<ol>
		<li>Although droplet diameter may vary slightly, ensure that gonads are not floating and are held in place by surface tension. Make sure that droplets do not touch each other or the side of the lid. Without the surface tension, the organs will grow onto the lid during the culture and flatten, thus distorting organ morphology.</li>
	</ol>
	</li>
	<li>Carefully place the culture dish lid containing the droplets upright onto the surface of the water in the large (100 mm) humidifier dish. Ensure there are no bubbles trapped underneath the bottom of the lid and that it is lying flat on the surface of the water. Do not invert the lid and be careful not to let any water touch the top of the lid where the droplets are located.</li>
	<li>To create a small, humidified chamber, immediately cover with the lid of the larger humidifier dish to enclose the gonad cultures. Act as quickly as possible to minimize any evaporation of media. Ensure that the smaller dish has room between it and the lid of the larger dish to move around freely; otherwise, condensation may make a seal and block air exchange to the tissue.</li>
	<li>Once two small lids are placed into the chamber, immediately place the chamber into the incubator. Incubate the droplet cultures for 48 hr in a humidified CO2 incubator at 37 &#176;C.</li>
</ol>
4. Polymerase Chain Reaction for Determining the Sex of Embryos
<ol>
	<li>Add the embryonic tails to bottom of a 1.5 ml&#160;microcentrifuge tube.</li>
	<li>Add to each tube 200 &#181;l&#160;of 50 mM NaOH.</li>
	<li>Place at 95 &#176;C for 15 min or until tissue is completely dissolved.</li>
	<li>Add 50 &#181;l&#160;of 1M Tris-HCl and vortex lightly and briefly.</li>
	<li>In a 1.5 ml&#160;microcentrifuge tube, make a fresh PCR master mix by multiplying each volume by the total number of samples: 0.5 &#181;l&#160;25 &#181;M Primer solution, 2.5 &#181;l&#160;10X Taq Buffer, 0.5 &#181;l&#160;10 mM dNTPs (nucleotides), 19.3 &#181;l&#160;nuclease-free water, 0.2 &#181;l&#160;DNA Taq polymerase. Mix well.</li>
	<li>Add 23 &#181;l&#160;of PCR master mix to PCR tubes containing 3 &#956;l&#160;lysate of digested tails.</li>
	<li>Flick tubes to mix them, then perform a short spin to bring the samples to the bottom of the tube.</li>
	<li>Run XY (Short) PCR program (Table 1).</li>
	<li>Load the samples (with 5x dye) and ladder in a 2.5% agarose gel in 1X TAE buffer.</li>
	<li>Run the agarose gel electrophoresis until XX and XY bands can be resolved.</li>
	<li>Image the gel with UV light and capture image.</li>
	<li>Analyze the X-chromosome-specific vs Y chromosome-specific bands (X chromosome=331 base pairs [bp] and XY=302 bp)13; XY (male) samples will have two bands of 331 and 302 bp, whereas XX (female) samples will appear as a single 331 bp band.</li>
</ol>
5. Whole Mount Organ Immunofluorescence
Day 1:
<ol>
	<li>Remove the gonads from the culture using a 1,000 &#181;l&#160;pipette with a cut-off tip. If desired, they may be placed in a dish of PBS to wash off media and reagents. Place into PBS in a 0.5 ml&#160;microcentrifuge tube. The smaller tube size will conserve reagents and make it easier to keep track of samples in the tube.
	<ol>
		<li>Be sure to keep control and treated samples, as well as XX and XY samples, in separate tubes and remove them from culture with separate sets of pipettes to avoid potential drug contamination.</li>
	</ol>
	</li>
	<li>Wash the gonads twice with PBS. From this point on, this protocol can use 1X PBS lacking calcium and magnesium. To wash them, allow the gonads to sink to the bottom of the tube (by gravity) and then remove the liquid above. Be careful not lose gonads by pipetting too close to them.</li>
	<li>Remove as much PBS as possible from the tube. Add 250 &#181;l&#160;of 4% paraformaldehyde in PBS containing 0.1% Triton X-100, and let incubate O/N at 4 &#176;C on a rocker. Alternatively, this fixation can be done for 2 hr at 4 &#176;C on a rocker. CAUTION! Paraformaldehyde is a toxic substance, so follow safety protocol when handling.</li>
</ol>
Day 2:
<ol start="4">
	<li>Rinse the gonads twice in 250 &#181;l&#160;PBTx at RT.</li>
	<li>Wash the gonads 3 times with 250 &#181;l&#160;PBTx for 10 min each at RT on a rocker.</li>
	<li>Block the gonads at least 1 hr in 250 &#181;l&#160;Blocking solution at RT on a rocker.</li>
	<li>Stain the gonads O/N at 4 &#176;C on a rocker in 250 &#181;l&#160;primary antibody diluted in Blocking Solution.</li>
</ol>
Day 3:
<ol start="8">
	<li>Rinse the gonads twice in 250 &#181;l&#160;Washing Solution.</li>
	<li>Wash the gonads 3 times with 250 &#181;l&#160;Washing Solution for 10 min each at RT on a rocker.</li>
	<li>Block the gonads 1 hr in 250 &#181;l&#160;Blocking Solution at RT on a rocker.</li>
	<li>Cover the microcentrifuge tube with aluminum foil to protect fluorescent secondary antibodies from light.</li>
	<li>Incubate the gonads 2-4 hr at RT on a rocker in 250 &#181;l&#160;secondary antibody diluted in Blocking Solution containing Hoechst 33342. Alternatively, perform secondary antibody and Hoechst 33342 incubation O/N at 4 &#176;C on rocker.</li>
	<li>Rinse the gonads twice in 250 &#181;l&#160;PBTx.</li>
	<li>Wash the gonads 3 times in 250 &#181;l&#160;PBTx for 10 min&#160;each at RT on a rocker.</li>
	<li>Using a cut-off pipette tip, transfer gonads onto a slide with minimal liquid. Remove excess liquid with pipette, but make sure that tissue does not dry out.</li>
	<li>Orient the gonads in desired fashion with forceps, and quickly add a drop of mounting media to mount the gonads on the slide. Make sure the mounting media coats all samples completely; it is critical to avoid letting samples dry out.</li>
	<li>Use coverslips (number 1.5 style) of appropriate size to gently cover samples. Let mounting media spread to contact the entire surface of coverslip. If necessary, very lightly press on the corners of the coverslip so that there are no large air bubbles.</li>
	<li>Seal the slides with clear nail polish around the edges to reduce evaporation of the mounting media. Store mounted slides in the dark at 4 &#176;C.</li>
</ol>

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The authors declare that they have no competing financial interests.

This study demonstrates an ex vivo whole organ droplet method that has many potential applications for studying fetal development. This technique can be used for multiple organs, and allows the researcher to address biological questions that are difficult to examine using in vivo approaches due to inaccessibility of embryos and potential embryonic lethality. This culture method has additional benefits over other in vitro approaches such as mammalian cell lines: whole organs can be used, therefo...

Organ regeneration in vivo in humans is very limited; therefore, tissue engineering, the development of tissues and organs from individual cells donated by a host, is becoming an attractive potential therapy for organ replacement. However, for this therapeutic strategy to be successful, factors and cellular interactions involved in morphogenesis of the organ must be thoroughly studied and well-understood. Due to the inability to study development of specific organs with traditional approaches, researchers have turned to alternative whole embryo or whole organ cultures. Kalaskar et al.1 have shown that ex vivo whole embryogenesis culture yields comparable results (in 58% of cultured embryos) to in utero development, suggesting that ex vivo culture methods are a feasible alternative for organogenesis studies.
An individualized organ culture system, such as this ex vivo droplet culture system, allows for whole organ analysis independent of systemic effects, while permitting manipulation of a specific signaling pathway or cellular interactions via addition of pharmacological reagents or antibodies. Traditionally, the study of fetal organ development has been limited to transgenic and knockout mouse technologies, in addition to pharmacological reagents delivered maternally. However, there are technical issues involving these techniques and treatments in vivo; most concerns revolve around the effects of influencing various organs simultaneously which often results in embryonic lethality. An additional concern of studies manipulating fetal development pharmacologically is the maternal effect of drugs on embryonic development in utero (e.g., maternal metabolism of the drug before it reaches the embryo) and if such reagents can pass through the placental barrier.
The whole organ culture technique described here was adapted from a protocol first described by Maatouk et al.2, in which whole fetal gonads are incubated in ex vivo upright droplet cultures. One significant advantage of culturing fetal gonads is that small-molecule inhibitors can readily access the whole organ by simple diffusion. DeFalco et al. have shown that utilizing this ex vivo droplet culture method in conjunction with small-molecule inhibitors can be used to study signaling processes and interactions occurring during gonad development3; these processes would be difficult to examine in vivo due to technical challenges (e.g., passage of drugs through the placenta or lethality of affecting multiple organs using genetic or pharmacological approaches).
The droplet culture is not only an improvement in certain aspects over in utero experimentation, but also it is an improvement over in vitro and ex vivo systems as well. The use of cell lines to study morphogenesis is extremely difficult because they lack the diverse cell types, lack critical extracellular matrix (ECM) components that permit the formation of organ architecture, and can exhibit artifacts in signaling cascades. Although tissue engineering has made significant improvements in creating scaffolds simulating ECM, the lack of knowledge with regard to which signals are required by each cell type during organogenesis makes it challenging to build an organ system in vitro. Other ex vivo systems have been previously established to study organogenesis, or more specifically morphogenesis, and have been very successful for live imaging of fetal organs in agar4, transwells5, filters6, and other scaffold matrices7,8. The advantage of the droplet culture system is that it allows the study of morphogenesis by providing the ability to utilize less reagents, which are often expensive, but also giving the organ surface tension, which is important for growth and signaling capabilities9.
In the mouse, initial testis morphogenesis takes place between embryonic (E) stages E11.5 and E13.5; these stages comprise the optimal time window for examining factors that influence sex-specific differentiation. Among the critical processes that occur during testis formation are the generation of testis cord architecture and the formation of a testis-specific vascular network. Utilizing this ex vivo whole organ droplet culture system, one is able to alter male-specific vascularization and inhibit testis morphogenesis through the use of a small-molecule inhibitor that blocks the activity of the receptors for vascular endothelial growth factor (VEGF); VEGF-mediated vascular remodeling is critical for testis development10-12. This technique can successfully be applied to other organs and can target specific time windows of development. Whole-mount organ imaging allows the visualization of vital structures as well as structural and cellular changes resulting from the administration of various inhibitors. Importantly, this system is advantageous in that the researcher can bypass potential confounding effects from maternal drug administration or systemic disruption during in vivo targeted gene strategies. Thus, this whole organ ex vivo droplet culture system can significantly improve the ability to understand the interactions and signaling which occur specifically within particular organs during fetal development.

<table><tbody><tr><td>Superfrost Plus Microscope Slides</td><td>Fisherbrand</td><td>12-550-15</td><td></td></tr><tr><td>Cover Glasses: Squares (22 mm x 22 mm, No. 1.5)</td><td>Fisherbrand</td><td>12-541B</td><td></td></tr><tr><td>Sally Hansen Xtreme Wear Nail Polish, Invisible</td><td>Sally Hansen</td><td></td><td></td></tr><tr><td>8-Strip 0.2 ml PCR Tubes &amp;amp; Detached Flat Caps</td><td>GeneMate</td><td>T3218-1</td><td></td></tr><tr><td>Pipetman L P1,000L, P200L, P20L, P10L, P2L</td><td>Gilson</td><td>FA10006M, FA10005M, FA10003M, FA10002M, FA10001M</td><td></td></tr><tr><td>Dumont #5 Forceps</td><td>FST</td><td>91150-20</td><td></td></tr><tr><td>Fine Scissors</td><td>FST</td><td>91460-11</td><td></td></tr><tr><td>Posi-Click 1.7 ml microcentrifuge tubes</td><td>Denville</td><td>C2170</td><td></td></tr><tr><td>Posi-Click 0.6 ml microcentrifuge tubes</td><td>Denville</td><td>C2176</td><td></td></tr><tr><td>10 &amp;mu;l SHARP Precision Barrier Tips</td><td>Denville</td><td>P1096FR</td><td></td></tr><tr><td>20 &amp;mu;l SHARP Precision Barrier Tips</td><td>Denville</td><td>P1121</td><td></td></tr><tr><td>200 &amp;mu;l SHARP Precision Barrier Tips</td><td>Denville</td><td>P1122</td><td></td></tr><tr><td>1,000 &amp;mu;l SHARP Precision Barrier Tips</td><td>Denville</td><td>P1126</td><td></td></tr><tr><td>1 ml syringe with 27 gauge needles</td><td>BD PrecisionGlide</td><td>309623</td><td></td></tr><tr><td>10 ml syringe</td><td>BD</td><td>305559</td><td></td></tr><tr><td>0.2 &amp;mu;M PES syringe filter</td><td>VWR</td><td>28145-501</td><td></td></tr><tr><td>Grade 3 Qualitative Filter Paper Standard Grade, circle, 185 mm</td><td>Whatman</td><td>1003-185</td><td></td></tr><tr><td>Primaria 35 mm Easy Grip Style Cell Culture Dish</td><td>Falcon/Corning</td><td>353801</td><td></td></tr><tr><td>Petri Dishes, Sterile (100 mm x 15 mm)</td><td>VWR</td><td>25384-088</td><td></td></tr><tr><td>New Brunswick Galaxy 14 S CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Eppendorf</td><td>CO14S-120-0000</td><td></td></tr><tr><td>Biosafety Cabinet</td><td>Nuare</td><td>NU-425-400</td><td></td></tr><tr><td>Mini-centrifuge&amp;nbsp;</td><td>Fisher Scientific</td><td>05-090-100</td><td></td></tr><tr><td>BioExpress GyroMixer Variable XL</td><td>GeneMate</td><td>R-3200-1XL</td><td></td></tr><tr><td>Mastercycler Pro Thermal Cycler with control panel</td><td>Eppendorf</td><td>950040015</td><td></td></tr><tr><td>SMZ445 stereomicroscope</td><td>Nikon</td><td>SMZ445</td><td></td></tr><tr><td>MultiImage Light Cabinet with AlphaEase Software</td><td>Alpha Innotech Corporation</td><td>Discontinued</td><td></td></tr><tr><td>Absolute 200 proof Ethanol</td><td>Fisher</td><td>BP2818-500</td><td></td></tr><tr><td>Triton X-100</td><td>Fisher</td><td>BP151-100</td><td></td></tr><tr><td>Sodium Phosphate (Dibasic MW 142) Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher</td><td>S374-1</td><td></td></tr><tr><td>Potassium Phosphate (Monobasic MW 136) KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P5379-1KG</td><td></td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Fisher</td><td>S671-3</td><td></td></tr><tr><td>Potassium Chloride (KCl)</td><td>Sigma-Aldrich</td><td>P3911-1KG</td><td></td></tr><tr><td>Magnesium Chloride (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma</td><td>M2393-100g</td><td></td></tr><tr><td>Calcium Chloride (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma</td><td>C5670-100g</td><td></td></tr><tr><td>Ambion Nuclease-Free Water</td><td>Life Technologies</td><td>AM9938&amp;nbsp;</td><td></td></tr><tr><td>XY PCR Primer&amp;nbsp;</td><td>IDT</td><td>N/A</td><td></td></tr><tr><td>Glacial Acetic Acid</td><td>Fisher</td><td>A38-500</td><td></td></tr><tr><td>Ethylenediamine Tetraacetic Acid (EDTA)</td><td>Fisher</td><td>BP2482-1</td><td></td></tr><tr><td>1% Ethidium bromide solution</td><td>Fisher</td><td>BP1302-10</td><td>Toxic</td></tr><tr><td>Agarose</td><td>GeneMate</td><td>E-3120-500</td><td></td></tr><tr><td>Sodium Hydroxide (NaOH)</td><td>Sigma-Aldrich</td><td>367176-2.5KG</td><td></td></tr><tr><td>Trizma Base</td><td>Sigma</td><td>T1503-1KG</td><td></td></tr><tr><td>dNTP Set, 100 mM Solutions</td><td>Thermo Scientific</td><td>R0182</td><td></td></tr><tr><td>DNA Choice Taq polymerase with 10x Buffer</td><td>Denville</td><td>CB-4050-3</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Fisher</td><td>O4042-500</td><td>Toxic</td></tr><tr><td>FluorMount-G</td><td>Southern Biotech</td><td>0100-01</td><td></td></tr><tr><td>Hydrogen Chloride (HCl)</td><td>Fisher</td><td>A144212</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA), powder, Fraction V, Heat shock isolation</td><td>Bioexpress</td><td>0332-100g</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)&amp;nbsp;</td><td>Life Technologies</td><td>11965-092</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS), triple 100 nm filtered</td><td>Fisher</td><td>03-600-511</td><td>Heat-inactivate before using</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/ml)</td><td>Life Technologies</td><td>15140-122</td><td>Use at 1:100</td></tr><tr><td>Dimethyl sulfoxide (DMSO), Hybri-max, sterile-filtered</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>VEGFR Tyrosine Kinase Inhibitor II - CAS 269390-69-4 - Calbiochem</td><td>EMD Millipore</td><td>676481</td><td></td></tr><tr><td>Rabbit Anti-Sox9 Antibody</td><td>Millipore</td><td>AB5535</td><td>Use at dilution: 1:4,000</td></tr><tr><td>Rat Anti-Mouse PECAM1 (CD31) Antibody</td><td>BD Pharmingen</td><td>553370</td><td>Use at dilution: 1:250</td></tr><tr><td>Rabbit Cleaved Caspase-3 (Asp175) Antibody</td><td>Cell Signaling</td><td>9661S</td><td>Use at dilution: 1:250</td></tr><tr><td>Rat E-cadherin / CDH1 Antibody (ECCD-2)</td><td>Life Technologies</td><td>13-1900</td><td>Use at dilution: 1:500</td></tr><tr><td>Hoechst 3342, trihydrochloride, trihydrate</td><td>Invitrogen (Molecular Probes)</td><td>H1399</td><td>Use at 2 ug/ml</td></tr><tr><td>Cy3 AffiniPure Donkey Anti-Rat IgG (H+L)</td><td>Jackson Immunoresearch</td><td>712-165-153</td><td>Use at dilution: 1:500</td></tr><tr><td>Alexa Fluor 647 AffiniPure Donkey Anti-Rat IgG (H+L)</td><td>Jackson Immunoresearch</td><td>712-605-153</td><td>Use at dilution: 1:500</td></tr><tr><td>Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 555 conjugate</td><td>Life Technologies</td><td>A31572</td><td>Use at dilution: 1:500</td></tr><tr><td>Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate</td><td>Life Technologies</td><td>A21206</td><td>Use at dilution: 1:500</td></tr><tr><td>Donkey anti-Rat IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate</td><td>Life Technologies</td><td>A21208</td><td>Use at dilution: 1:500</td></tr><tr><td>Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 647 conjugate</td><td>Life Technologies</td><td>A31573</td><td>Use at dilution: 1:500</td></tr></tbody></table>

using ex vivo upright droplet cultures of whole fetal organs to study developmental processes during mouse organogenesis

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop within the uterus. A newly developed ex vivo upright droplet culture method provides an attractive alternative to studies performed in utero. The ex vivo droplet culture provides the ability to examine and manipulate cellular interactions and diverse signaling pathways through use of various blocking and activating compounds; additionally, the effects of various pharmacological reagents on the development of specific organs can be studied without unwanted side effects of systemic drug delivery in utero. As compared to other in vitro systems, the droplet culture not only allows for the ability to study three-dimensional morphogenesis and cell-cell interactions, which cannot be reproduced in mammalian cell lines, but also requires significantly less reagents than other ex vivo and in vitro protocols. This paper demonstrates proper mouse fetal organ dissection and upright droplet culture techniques, followed by whole organ immunofluorescence to demonstrate the effectiveness of the method. The ex vivo droplet culture method allows the formation of organ architecture comparable to what is observed in vivo and can be utilized to study otherwise difficult-to-study processes due to embryonic lethality in in vivo models. As a model application system, a small-molecule inhibitor will be utilized to probe the role of vascularization in testicular morphogenesis. This ex vivo droplet culture method is expandable to other fetal organ systems, such as lung and potentially others, although each organ must be extensively studied to determine any organ-specific modifications to the protocol. This organ culture system provides flexibility in experimentation with fetal organs, and results obtained using this technique will help researchers gain insights into fetal development.

The ex vivo droplet culture allows one to manipulate whole organs, such as the gonad, to study cellular interactions and dynamics. Figure 1 demonstrates in a step-wise fashion how to prepare an E11.5 gonadal droplet culture. The first steps in the culture protocol include the initial removal of the embryo-containing uterus from the mother mouse (Figure 1A and 1B). After removal of the uterus from the mother, the uterine wall is cut and the embryos are liberated from the yolk sac...

Watch this Scientific Journal Video about Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis at JoVE.com

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

The&#160;ex vivo&#160;upright droplet culture is an alternative to current&#160;in vitro&#160;and&#160;in vivo&#160;experimental techniques. This protocol is easy to perform and requires smaller amounts of reagent, while permitting the ability to manipulate and study fetal vascularization, morphogenesis, and organogenesis.

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop ...

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Research

JoVE Journal

Developmental Biology

12.4K Views.  Cincinnati Children's Hospital Medical Center. The ex vivo upright droplet culture is an alternative to current in vitro and in vivo experimental techniques. This protocol is easy to perform and requires smaller amounts of reagent, while permitting the ability to manipulate and study fetal vascularization, morphogenesis, and organogenesis.

Video: Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Organ Culture and Whole Mount Immunofluorescence Staining of Mouse Wolffian Ducts

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an integral part of the male reproductive tract, is responsible for sperm storage, maturation, and transport. The adult epididymis is a highly coiled tube that develops from a simple and straight embryonic precursor known as Wolffian duct (WD). Proper coiling of the epididymis is essential for male fertility, as sperm in the testis are unable to fertilize an oocyte. However, the mechanism responsible for epididymal development and coiling remains unclear, partially due to the lack of whole organ culture and imaging methods. In this study, we describe an in vitro culture system and whole mount immunofluorescence protocol to better visualize the process of WD coiling and development, which may also be applied to study other tubular organs.

We present a method for isolation and culture of the mouse Wolffian duct (WD). We also demonstrate a detailed procedure for whole mount immunostaining of cultured/freshly isolated WDs with fluorescently tagged antibodies. Together, these techniques enable the study of WD development, coiling, and differentiation.

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an ...

Three-Dimensional Imaging of Organoids to Study Primary Ciliogenesis During ex vivo Organogenesis

Organoids are stem cell-derived three-dimensional structures that reproduce ex vivo the complex architecture and physiology of organs. Thus, organoids represent useful models to study the mechanisms that control stem cell self-renewal and differentiation in mammals, including primary ciliogenesis and ciliary signaling. Primary ciliogenesis is the dynamic process of assembling the primary cilium, a key cell signaling center that controls stem cell self-renewal and/or differentiation in various tissues. Here we present a comprehensive protocol for the immunofluorescence staining of cell lineage and primary cilia markers, in whole-mount mouse mammary organoids, for light sheet microscopy. We describe the microscopy imaging method and an image processing technique for the quantitative analysis of primary cilium assembly and length in organoids. This protocol enables a precise analysis of primary cilia in complex three-dimensional structures at the single cell level. This method is applicable for immunofluorescence staining and imaging of primary cilia and ciliary signaling in mammary organoids derived from normal and genetically modified stem cells, from healthy and pathological tissues, to study the biology of the primary cilium in health and disease.

Three-Dimensional Imaging of Organoids to Study Primary Ciliogenesis During ex vivo Organogenesis

Stem cell-derived organoids facilitate the analysis of molecular and cellular processes that regulate stem cell self-renewal and differentiation during organogenesis in mammalian tissues. Here we present a protocol for the analysis of the biology of the primary cilium in mouse mammary organoids.

Organoids are stem cell-derived three-dimensional structures that reproduce ex vivo the complex architecture and physiology of organs. Thus, organoids ...

Genetics

Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony forming units (CFU-Es). After CFU-Es, cells are morphologically recognizable and generally termed terminal erythroblasts. One of the challenges for the study of terminal erythropoiesis is the lack of experimental approaches to dissect gene functions in a chronological manner. In this protocol, we describe a unique strategy to determine gene functions in the early and late stages of terminal erythropoiesis. In this system, mouse fetal liver TER119 (mature erythroid cell marker) negative erythroblasts were purified and transduced with exogenous expression of cDNAs or small hairpin RNAs (shRNAs) for the genes of interest. The cells were subsequently cultured in medium containing growth factors other than erythropoietin (Epo) to maintain their progenitor stage for 12 hr while allowing the exogenous cDNAs or shRNAs to express. The cells were changed to Epo medium after 12 hr to induce cell differentiation and proliferation while the exogenous genetic materials were already expressed. This protocol facilitates analysis of gene functions in the early stage of terminal erythropoiesis. To study late stage terminal erythropoiesis, cells were immediately cultured in Epo medium after transduction. In this way, the cells were already differentiated to the late stage of terminal erythropoiesis when the transduced genetic materials were expressed. We recommend a general application of this strategy that would help understand detailed gene functions in different stages of terminal erythropoiesis.

We present an in vitro mouse fetal liver erythroblast culture system that dissects the early and late stages of terminal erythropoiesis. This system facilitates functional analysis of specific genes in different developmental stages.

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony ...

Immunology and Infection

Recapitulating Suckling-to-Weaning Transition In Vitro using Fetal Intestinal Organoids

At the end of the suckling period, many mammalian species undergo major changes in the intestinal epithelium that are associated with the capability to digest solid food. This process is termed suckling-to-weaning transition and results in the replacement of neonatal epithelium with adult epithelium which goes hand in hand with metabolic and morphological adjustments. These complex developmental changes are the result of a genetic program that is intrinsic to the intestinal epithelial cells but can, to some extent, be modulated by extrinsic factors. Prolonged culture of mouse primary intestinal epithelial cells from late fetal period, recapitulates suckling-to-weaning transition in vitro. Here, we describe a detailed protocol for mouse fetal intestinal organoid culture best suited to model this process in vitro. We describe several useful assays designed to monitor the change of intestinal functions associated with suckling-to-weaning transition over time. Additionally, we include an example of an extrinsic factor that is capable to affect suckling-to-weaning transition in vivo, as a representation of modulating the timing of suckling-to-weaning transition in vitro. This in vitro approach can be used to study molecular mechanisms of the suckling-to-weaning transition as well as modulators of this process. Importantly, with respect to animal ethics in research, replacing in vivo models by this in vitro model contributes to refinement of animal experiments and possibly to a reduction in the use of animals to study gut maturation processes.

This protocol describes how to mimic suckling-to-weaning transition in vitro using mouse late fetal intestinal organoids cultured for 30 days.

At the end of the suckling period, many mammalian species undergo major changes in the intestinal epithelium that are associated with the capability to ...

Optimization of Renal Organoid and Organotypic Culture for Vascularization, Extended Development, and Improved Microscopy Imaging

Embryonic kidney organotypic cultures, and especially pluripotent stem cell-derived kidney organoids, are excellent tools for following developmental processes and modelling kidney disease. However, the models are limited by a lack of vascularization and functionality. To address this, an improved protocol for the method of xenografting cells and tissues to the chorioallantoic membrane (CAM) of an avian embryo to gain vascularization and restoration of blood flow was developed. The grafts are overlaid with custom-made minireservoirs that fix the samples to the CAM and supply them with culture medium that protects the grafts from drying. The improved culture method allows xenografts to grow for up to 9 days. The manuscript also describes how to provide optimal conditions for long-term confocal imaging of renal organoids and organotypic cultures using the previously published Fixed Z-Direction (FiZD) method. This method gently compresses an embryonic organ or organoid between a glass coverslip and membrane in a large amount of medium and provides excellent conditions for imaging for up to 12 days. Together, these methods allow vascularization and blood flow to renal organoids and organotypic kidney cultures with improved confocal imaging. The methods described here are highly beneficial for studying fundamental and applied functions of kidneys ex vivo. Both methods are applicable to various types of tissues and organoids.

This work describes two methods for studying organ development, an improved xenotransplantation setup on chorioallantoic membrane (CAM) from avian embryos that allows for vascularization of cultured embryonic organs and organoids and a novel fixed z-direction organ culture method with modified experimental conditions that allows for high-resolution time-lapse confocal imaging.

Embryonic kidney organotypic cultures, and especially pluripotent stem cell-derived kidney organoids, are excellent tools for following developmental ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. Platforms have been recently developed for highly robust and prolonged ex utero culture of mouse embryos from egg-cylinder stages until advanced organogenesis. These platforms enable appropriate and faithful development of pregastrulating embryos (E5.5) until the hind limb formation stage (E11). Late gastrulating embryos (E7.5) are grown in rotating bottles in these settings, while extended culture from pregastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle cultures. In addition, sensitive regulation of O2 and CO2 concentration, gas pressure, glucose levels, and the use of a specific ex utero culture medium are critical for proper embryo development. Here, a detailed step-by-step protocol for extended ex utero mouse embryo culture is provided. The ability to grow normal mouse embryos ex utero from gastrulation to organogenesis represents a valuable tool for characterizing the effect of different experimental perturbations during embryonic development.

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

An enhanced platform for whole-embryo culture allows continuous and robust ex utero development of postimplantation mouse embryos for up to six days, from pregastrulation stages until advanced organogenesis. In this protocol, we detail the standard procedure for successful embryo culture using static plates and rotating bottle systems.

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Biology

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...

Neuroscience

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

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Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System