Name: using ex vivo upright droplet cultures of whole fetal organs to study developmental processes during mouse organogenesis
Uploaded: 2015-10-21T15:00:00
Description: 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

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 Institutiona...

<ol>
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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.

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			<td>Superfrost Plus Microscope Slides</td>
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			<td>Sally Hansen Xtreme Wear Nail Polish, Invisible</td>
			<td>Sally Hansen</td>
			<td>N/A</td>
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			<td>8-Strip 0.2 mL PCR Tubes &#38; Detached Flat Caps</td>
			<td>GeneMate</td>
			<td>T3218-1</td>
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			<td>Pipetman L P1000L, P200L, P20L, P10L, P2L</td>
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			<td>FA10006M, FA10005M, FA10003M, FA10002M, FA10001M</td>
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			<td>Dumont #5 Forceps</td>
			<td>FST</td>
			<td>91150-20</td>
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			<td>Fine Scissors</td>
			<td>FST</td>
			<td>91460-11</td>
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			<td>Posi-Click 1.7 ml microcentrifuge tubes</td>
			<td>Denville</td>
			<td>C2170</td>
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			<td>Posi-Click 0.6 ml microcentrifuge tubes</td>
			<td>Denville</td>
			<td>C2176</td>
			<td></td>
		</tr>
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			<td>10 &#956;l SHARP Precision Barrier Tips</td>
			<td>Denville</td>
			<td>P1096FR</td>
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			<td>20 &#956;l SHARP Precision Barrier Tips</td>
			<td>Denville</td>
			<td>P1121</td>
			<td></td>
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			<td>200 &#956;l SHARP Precision Barrier Tips</td>
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			<td>P1122</td>
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		<tr>
			<td>1000 &#956;l SHARP Precision Barrier Tips</td>
			<td>Denville</td>
			<td>P1126</td>
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			<td>1 ml syringe with 27gauge needles</td>
			<td>BD PrecisionGlide</td>
			<td>309623</td>
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			<td>10 ml syringe</td>
			<td>BD</td>
			<td>305559</td>
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			<td>0.2 &#956;M PES syringe filter</td>
			<td>VWR</td>
			<td>28145-501</td>
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			<td>Grade 3 Qualitative Filter Paper Standard Grade, circle, 185 mm</td>
			<td>Whatman</td>
			<td>1003-185</td>
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			<td>Primaria 35mm Easy Grip Style Cell Culture Dish</td>
			<td>Falcon/Corning</td>
			<td>353801</td>
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			<td>Petri Dishes, Sterile (100 mm x 15 mm)</td>
			<td>VWR</td>
			<td>25384-088</td>
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			<td>New Brunswick Galaxy 14 S CO2 Incubator</td>
			<td>Eppendorf</td>
			<td>CO14S-120-0000</td>
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			<td>Biosafety Cabinet</td>
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			<td>NU-425-400</td>
			<td></td>
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			<td>Mini-centrifuge&#160;</td>
			<td>Fisher Scientific</td>
			<td>05-090-100</td>
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			<td>BioExpress GyroMixer Variable XL</td>
			<td>GeneMate</td>
			<td>R-3200-1XL</td>
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			<td>Mastercycler Pro Thermal Cycler with control panel</td>
			<td>Eppendorf</td>
			<td>950040015</td>
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			<td>SMZ445 stereomicroscope</td>
			<td>Nikon</td>
			<td>SMZ445</td>
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			<td>MultiImage Light Cabinet with AlphaEase Software</td>
			<td>Alpha Innotech Corporation</td>
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			<td>Fisher</td>
			<td>BP2818-500</td>
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			<td>Triton X-100</td>
			<td>Fisher</td>
			<td>BP151-100</td>
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			<td>Sodium Phosphate (Dibasic MW 142) Na2HPO4</td>
			<td>Fisher</td>
			<td>S374-1</td>
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			<td>Potassium Phosphate (Monobasic MW 136) KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P5379-1KG</td>
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			<td>Sodium Chloride (NaCl)</td>
			<td>Fisher</td>
			<td>S671-3</td>
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			<td>Potassium Chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
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			<td>Magnesium Chloride (MgCl2)</td>
			<td>Sigma</td>
			<td>M2393-100g</td>
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			<td>Sigma</td>
			<td>C5670-100g</td>
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			<td>Life Technologies</td>
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			<td>1% Ethidium bromide solution</td>
			<td>Fisher</td>
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			<td>Sodium Hydroxide (NaOH)</td>
			<td>Sigma-Aldrich</td>
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			<td>DNA Choice Taq polymerase with 10x Buffer</td>
			<td>Denville</td>
			<td>CB-4050-3</td>
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			<td>Paraformaldehyde</td>
			<td>Fisher</td>
			<td>O4042-500</td>
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			<td>FluorMount-G</td>
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			<td>Hydrogen Chloride (HCl)</td>
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			<td>Bovine Serum Albumin (BSA), powder, Fraction V, Heat shock isolation</td>
			<td>Bioexpress</td>
			<td>0332-100g</td>
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			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)&#160;</td>
			<td>Life Technologies</td>
			<td>11965-092</td>
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			<td>Fetal Bovine Serum (FBS), triple 100-nm filtered</td>
			<td>Fisher</td>
			<td>03-600-511</td>
			<td>Heat-inactivate before using</td>
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			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td>Use at 1:100</td>
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		<tr>
			<td>Dimethyl sulfoxide (DMSO), Hybri-max, sterile-filtered</td>
			<td>Sigma</td>
			<td>D2650</td>
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		</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 2ug/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 ...

Research

JoVE Journal

Developmental Biology

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Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

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