We would like to thank Dr. Julie Gordon and Dr. Nancy Manley for their helpful advice with the culture technique. This work has been supported by the Children&#8217;s Glaucoma Foundation and Sharon-Stewart Aniridia Research Trust.

Mouse embryo culture:
All experimental procedures were conducted in strict accordance with National Institutes of Health guidelines following protocol # A2010 07-119, which was reviewed and approved by the University of Georgia Institutional Animal Care and Use Committee, which maintains continued regulatory oversight.
1. Preparation of Culture Media
<ol>
	<li>Prepare culture medium using commercially available stem cell media and supplements in the following amounts: KnockOut DMEM, KnockOut Serum Replacement (KSR) (10%), N-2 Supplement (1x), Albumin, from Bovine Serum (2%), Penicillin (50 IU/ml), Streptomycin (50 &#956;g/ml) and Amphotericin-B (1.25 &#956;g/ml). The culture medium prepared is similar to that previously described15 with the following changes: addition of anti-mycotics and using twice the concentration of antibiotics (for details of specific reagents and equipment,&#160;see&#160;List of Materials). 
	Note: Antibiotic concentration as previously used (Moore-Scott, et al.15) was sufficient for embryo cultures carried up to 24 hr&#160;time period. However, when culture was continued beyond 24 hr, we experienced contamination of the culture and this was successfully controlled by doubling the antibiotic concentration.</li>
	<li>Store the media components at 4 &#176;&#8203;C or at -20 &#176;&#8203;C as per manufacturer recommendations. KSR and N-2 Supplement should be stored in aliquots at -20 &#176;&#8203;C to avoid repeated freezing and thawing. KnockOut DMEM once opened should be utilized within 30 days as per manufacturer&#8217;s recommendation. Prior to use, warm the components in a water bath to 37 &#176;&#8203;C.</li>
	<li>Prepare the media under sterile conditions. Disinfect the surface of all the equipment and bottles containing the media components with 70% alcohol spray before placing them in the culture hood.</li>
	<li>The media components can be mixed either in a sterile beaker or directly in the filter system (Corning). First add the albumin powder to the KnockOut DMEM. Mix thoroughly by gently shaking until the albumin completely dissolves. Then add the N-2 Supplement, KSR and the antibiotics and mix thoroughly using a pipettor. Then filter sterilize the media using a 0.22 &#181;m pore size filter with vacuum suction. Dispense the media into 50 ml conical tubes and store at 4 &#176;&#8203;C until used. The media once prepared should be used within 4-5 days for culture.</li>
</ol>
2. Preparation of Culture Equipment
<ol>
	<li>Sterilize the glass culture bottles and rubber corks. Wrap the culture bottles in an aluminum foil and place the rubber bottle corks in a water beaker and sterilize by autoclaving.</li>
	<li>Sterilize the culture chamber (Precision Incubator Unit) with 70% alcohol spray and warm to 37 &#176;C before starting the culture. The culture chamber is equipped with a rolling disc at the center which holds the culture bottles. Gas flow into the chamber is regulated by a pressure gauge and the flow rate can be adjusted by observing the outflow of air bubbles through water in a glass tube at the end. This rolling bottle culture apparatus is a modified version of the original apparatus introduced by New and Cockroft17.</li>
	<li>Connect a gas cylinder containing a gas mixture of 95% O2 / 5% CO2 to the culture chamber and adjust the gas flow to &#8220;one air bubble per second&#8221; which gives a flow rate of 50 cc/min and ensures adequate oxygen supply to the culture embryos.</li>
</ol>
3. Mouse Embryo Collection and Preparation for Culture
<ol>
	<li>To obtain wild type mouse embryos, we utilized mice of C57BL/6J and CD-1 (Charles River Laboratories) genetic background strains.</li>
	<li>Check the female mice for vaginal plugs every day morning. The noon on the day of finding the vaginal plug should be considered as E0.5 dpc (days post coitus).</li>
	<li>Collect mouse embryos at E10.5 dpc by euthanizing the pregnant mice following standard protocols. Mice were euthanized using a CO2 inhalation device as specified by the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (2013). To insure death, cervical dislocation was performed after exposure to CO2.</li>
	<li>Spray 70% ethanol on the ventral abdominal surface to avoid sticking of abdominal hair to the instruments. Then open the abdominal cavity ventrally using a sharp-blunt operating scissors&#160;and a pair of 4-3/4 in&#160;microdissecting forceps&#160;to locate the uterus. Using a 4 in&#160;microdissecting forceps&#160;lift the entire uterus and separate it from the body by cutting with a light operating scissors&#160;at the uterine body and at the tips of the uterine horns.</li>
	<li>Quickly rinse the entire uterus in 1x PBS warmed to 37 &#176;C to remove any blood sticking to the uterus and immediately place in DMEM warmed to 37 &#176;C in a Petri dish. Sterilize the instruments by 70% ethanol spray before further use. 
	Note: Preheating the solutions including the PBS, DMEM and the culture medium and maintaining them at 37 &#176;&#8203;C during the entire procedure is recommended.</li>
	<li>Under a dissecting microscope, the uterus should be segmentally dissected with a light operating scissors. This results in small openings on either side of the segmented uterus through which a pair of modified (blunted ends) microdissecting tweezers&#160;can be gently inserted to widen the opening. This permits the exposure of the placental decidua, which can then be removed by gently tearing with a pair of microdissecting tweezers to expose the parietal yolk sac (PYS) with the Reichert&#8217;s membrane. Using one edge of the tweezers gently pierce the Reichert&#8217;s membrane along with the PYS and separate from the underlying visceral yolk sac (VYS) layer to expose the embryos with intact VYS. The ectoplacental cone and the trophoectoderm derivatives can be removed either with the placental decidua or with the Reichert&#8217;s membrane. Care must be taken to avoid rupture of the VYS as the embryos pop out immediately.</li>
	<li>The embryos with intact VYS should then be transferred to a Petri dish containing the culture medium warmed to 37 &#176;&#8203;C using a plastic transfer pipette. 
	Note:&#160;Transfer to culture medium immediately after separation from uterus helps for better development of the embryo in culture.</li>
	<li>A small opening should be made in the yolk sac with a pair of microdissecting tweezers avoiding major blood vessels. A sharp pair of tweezers can be used to make the opening by gently piercing in an area adjacent to the head region. Alternatively two pairs of blunt tweezers can be used to hold the yolk sac and gently tear to make a small opening. Then gently widen the opening by expanding with the tweezers to a size just enough to fit the embryonic head. The amniotic membrane which is closely wrapping the embryo should be gently held away from the body and torn using a pair of tweezers. The embryonic head and later the whole embryo should be gently exteriorized from the yolk sac while maintaining the integrity of the embryonic vasculature with that of the yolk sac15. 
	Note:&#160;Any damage to the yolk sac vasculature can potentially affect the development of the embryo in culture. Such embryos with damaged vasculature should not be used for culture and can be used for staging the embryos which can later be discarded.</li>
	<li>Embryonic staging criteria: Examine the embryos under the dissecting microscope and group by morphological criteria including body and head size, limb and eye morphology and stage by counting the number of somite(s)&#160;for at least two embryos from each group. 
	Note:&#160;Usually embryos obtained from the same litter show differences in development in utero and differ in body size, morphological features and number of somites. However, we found that embryos grouped by our morphological criteria usually exhibited similar somite number (&#177;1). For this reason, it is not required to count somites for each embryo as this would delay the time for starting the culture. 
	Note:&#160;Do not use the embryos that were used to count the somites for culture. Count the somites after starting the culture for other embryos in order to avoid the time delay in starting the culture. The embryos that were delayed to culture after separation from the uterus usually exhibit poor development.</li>
	<li>Transfer the embryos immediately to a Petri dish with fresh culture medium warmed to 37 &#176;&#8203;C and take it to the culture hood where the embryos should again be transferred to a Petri dish with sterile culture media before putting them into the culture bottles with medium to start the culture. 
	Note:&#160;The multiple media transfers for the embryos before culture helps in preventing contamination as the different steps of embryo collection and dissection were performed under a dissecting microscope which was not installed in the culture hood. 
	Note:&#160;When the litter size is large (&#62;12 embryos), process half the embryos and start the culture or put them in a dish with culture medium and place it in a CO2 incubator at 37 &#176;&#8203;C.&#160;When embryos were left outside for longer periods (&#62;35-40 min) we observed the heartbeat to slow down and this will affect the later development in the culture.</li>
</ol>
4. Culturing Mouse Embryos
<ol>
	<li>Open the autoclaved culture bottles in the culture hood and properly label them. Transfer 3 ml of culture medium warmed to 37 &#176;&#8203;C to each of the bottles and then the mouse embryos should be transferred gently into the culture bottles using sterile plastic transfer pipettes. The culture bottles should then be capped with the rubber bottle corks which help to hold the culture bottles to the rolling disc in the culture apparatus. 
	Note:&#160;The culture bottles with the media can be prepared and placed on the rolling disc of the culture apparatus before euthanizing the mice. This shortens the time for transfer of embryos into the culture bottles. 
	Note:&#160;Mouse embryos can be cultured individually or as cocultures with two or three embryos in the same culture bottle.</li>
	<li>The culture bottles should be aseptically carried to the culture apparatus at 37 &#176;&#8203;C and tightly hook them to the holes on the rolling disc with the rubber corks. Turn on the rolling disc to rotate at a constant speed of 35 rotations per minute (rpm), which enables the free floatation of the embryos in the media and also helps in free gas exchange by the embryonic tissue. The gas from the cylinder connected to the culture chamber flows through the rolling disc and the rubber corks into the culture bottles.</li>
	<li>Culture the embryos for 16-40 hr&#160;and regularly check for the gas outflow and culture chamber temperature. 
	Note:&#160;Mouse embryo manipulation in culture: Let the embryos get adapted to the culture conditions for at least 30 min. Then embryos that perform poorly and showing feeble heartbeat can be discarded and the remaining embryos can be utilized for further manipulation studies. Embryo manipulations such as electroporation5,9,16, microinjections18, bead implantation16, drug treatment and other procedures can be performed at this time. We successfully performed implantation of affi-gel agarose beads treated with certain signaling molecules to study their role in early eye development. The embryos after manipulations can be returned back in a fresh medium and continued in culture.</li>
	<li>Replace the culture media totally with fresh media at specific intervals after 9-10 hr&#160;and 18-19 hr&#160;of culture when the culture was continued for 40 hr. 
	Note:&#160;Culture media replacement may not be required if the culture is stopped by 16-18 hr.</li>
	<li>Measures of success in culture: Embryonic survival in culture should be determined by visible heartbeat and blood circulation in the body while embryonic growth in culture should be assessed by increase in body size, somite number and morphological development of head, limbs, heart and eyes.</li>
	<li>In utero developed embryos at E11.0 dpc (~40-41 s) and E12.0 dpc (~49-50 s) should be used as controls for comparing embryos cultured for 16-18 hr&#160;and 38-40 hr, respectively.</li>
	<li>Embryonic development at different time points during culture and for in utero stages can be captured under dissecting microscope. Spot imaging software was utilized to capture the images and later processed using image processing software.</li>
</ol>

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We do not have any competing financial interests.

Mid-gestation stage mouse embryos were cultured in a serum-free culture media in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C. Embryo development ex utero was critically dependent on multiple factors at each step during the procedure from the time the uterus is isolated from the euthanized mice to the completion of the culture (Figure 1). The most important factor that influenced the development was the time taken to start the ...

In vitro culture methods utilizing whole embryos are well suited to study signaling mechanisms involved in embryonic organogenesis that are otherwise difficult to access in utero. Whole embryos provide the tissue integrity and support appropriate for the tissue interactions that are crucial for timely occurrence of signaling mechanisms essential for different cellular processes during organogenesis. While whole embryo cultures provide a platform for a plethora of applications such as transplantation studies, genetic and tissue manipulations, bead implantation studies, toxicological studies, etc., currently utilized embryo culture systems are mainly dependent on serum for proper growth and maintenance of the embryos in the culture1-9.
Serum has been utilized as one of the major components ranging from 10-100% of the culture media6-8,10,11.&#160; However, the composition of serum is not well defined and can differ from animal to animal and each time the serum is collected. While laboratory preparation of serum is time consuming and involves stringent procedures, serum procured commercially exhibits considerable variability among different lots and raises experimental costs. Added to these, the serum may contain unknown factors such as growth factors, hormones, or other proteins, which can potentially affect the outcome of certain experiments, especially those that involve the study of signaling molecules important in tissue interactions. Studies have shown that addition of serum to culture can potentially alter the intracellular levels of certain signaling molecules such as cyclic adenosine monophosphate (cAMP) and proteins involved in mitogenic signaling and phosphoinositide 3 (PI 3)-kinase signaling pathways12-14. Contrary to these, a serum-free culture system provides the advantages of antigen free environment, abstinence from biologically active enzymes that can alter the cellular processes and enables consistency among experiments.
In the present study, we utilized a serum-free culture medium prepared from commercially available stem cell media supplements to culture mid-gestation stage whole embryos in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C15,16.&#160; Mouse embryos cultured for 16 to 40 hr&#160;under these defined conditions exhibited progression in morphological development of overall embryonic body and different structures such as the heart, limbs, brain and eyes indicating appropriate levels of cellular proliferation, migration, differentiation and tissue interactions. Molecular analysis of the embryonic development in the culture for one of the complex organ systems such as the eye revealed the ocular development to be consistent with that observed in ocular tissue in embryos developing in utero (Kalaskar and Lauderdale, in preparation). Thus we show that mouse embryos cultured at mid-gestation stage, exhibit progressive growth and morphological development comparable to that observed in embryos developing in utero.

<table border="0" cellpadding="0" cellspacing="0" width="634">
	<tbody>
		<tr>
			<td>KnockOut DMEM</td>
			<td>Invitrogen</td>
			<td>10829-018</td>
			<td></td>
		</tr>
		<tr>
			<td>KnockOut Serum Replacement</td>
			<td>Invitrogen</td>
			<td>10828-028</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2 Supplement</td>
			<td>Invitrogen</td>
			<td>17502-048</td>
			<td>Stock: 100x</td>
		</tr>
		<tr>
			<td>Albumin, from Bovine Serum</td>
			<td>Sigma</td>
			<td>A9418-50G</td>
			<td>Stock: 100%</td>
		</tr>
		<tr>
			<td>Antibiotic - Antimycotic Solution</td>
			<td>Cellgro</td>
			<td>30-004-Cl</td>
			<td>Stock: Penicillin (10000 IU/ml); Streptomycin (10000 &#181;g/ml; Amphotericin (250&#181;g/ml)&#160;</td>
		</tr>
		<tr>
			<td>DMEM&#160;&#160;</td>
			<td>Cellgro</td>
			<td>15-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision Incubator Unit</td>
			<td>B.T.C. Engineering Milton Cambridge England</td>
			<td>Id.No. 840-374</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Bottles for Rotating Unit</td>
			<td>B.T.C. Engineering</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone Rubber Cork</td>
			<td>B.T.C. Engineering</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>95% O2/5% CO2 Cylinder</td>
			<td>AirGas Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stemi SV11 Apo Dissecting Microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stemi SV6 Microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Water Jacketed Incubator</td>
			<td>Forma Scientific</td>
			<td>Model: 3110</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture Hood</td>
			<td>Nuaire Biological Safety Cabinets Class II TypeA2</td>
			<td>Model: Nu-425-600</td>
			<td></td>
		</tr>
		<tr>
			<td>Water Bath</td>
			<td>Fisher Scientific</td>
			<td>IsoTemp205</td>
			<td></td>
		</tr>
		<tr>
			<td>Weigh Balance</td>
			<td>Mettler Toledo&#160;</td>
			<td>AG285</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Tube &#8211; 50ml</td>
			<td>Corning</td>
			<td>430291</td>
			<td></td>
		</tr>
		<tr>
			<td>Light Operating Scissors</td>
			<td>Roboz</td>
			<td>RS-6702</td>
			<td></td>
		</tr>
		<tr>
			<td>Operating Sharp-Blunt Scissors&#160;</td>
			<td>Roboz</td>
			<td>RS-6812</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Dissecting Forceps &#8211; 4&#8221;</td>
			<td>Roboz</td>
			<td>RS-5211</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Dissecting Forceps - Hudson (cWALD) &#8211; 4-3/4&#8221;</td>
			<td>Roboz</td>
			<td>RS-5237</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Dissecting Tweezers (5/45)</td>
			<td>Roboz</td>
			<td>RS-5005</td>
			<td>Modified - Sharp ends were made blunt</td>
		</tr>
		<tr>
			<td>Micro Dissecting Tweezers (5)</td>
			<td>Roboz</td>
			<td>RS-5060</td>
			<td>Modified - Sharp ends were made blunt</td>
		</tr>
		<tr>
			<td>Micro Dissecting Tweezers (55)</td>
			<td>Roboz</td>
			<td>RS-5063</td>
			<td>Modified - Sharp ends were made blunt</td>
		</tr>
		<tr>
			<td>Instrument Tray</td>
			<td>Roboz</td>
			<td>RT-1401S</td>
			<td></td>
		</tr>
		<tr>
			<td>Instrument Tray Lid</td>
			<td>Roboz</td>
			<td>RT-1401L</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish-100mm</td>
			<td>Fisher Scientific</td>
			<td>087571Z</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish-60mm</td>
			<td>Fisher Scientific</td>
			<td>0875713A</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish-35mm</td>
			<td>Fisher Scientific</td>
			<td>0875711YZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter System ( 0.22um Cellulose Acetate)-150ml</td>
			<td>Corning</td>
			<td>431153</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter System ( 0.22um Cellulose Acetate)-250ml</td>
			<td>Corning</td>
			<td>430756</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter System ( 0.22um Cellulose Acetate)-500ml</td>
			<td>Corning</td>
			<td>430758</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet-aid Pipetter</td>
			<td>Drummond Scientific Co.</td>
			<td>D57849</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipette-10ml</td>
			<td>VWR</td>
			<td>89130-898</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Serological Pipette-25ml</td>
			<td>Corning</td>
			<td>4251</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer Pipette - 7.7ml</td>
			<td>Thermo Scientific</td>
			<td>202-20S</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Development of mouse embryos ex utero depends on multiple factors starting from the time the uterus is isolated from the body to the time the embryos are cultured. As depicted in Figure 1, the procedure involves a series of steps including, separation of the gravid uterus from the body (Figure 1A), isolation of the embryos with intact yolk sac (Figure 1B), exteriorization of the embryos from the yolk sac (Figure 1C) and culturing the embryos in ...

Watch this Scientific Journal Video about Mouse Embryonic Development in a Serum-free Whole Embryo Culture System at JoVE.com

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

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 ...

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25.7K Views.  University of Georgia. 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 exhibit morphological development comparable to embryos developing in utero.

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

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

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 ...

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development and growth of mammalian embryos are critically dependent on the function of the placenta, WEC technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages during embryonic day (E) 6.5-E12.5 in the mouse or E8.5-E14.5 in the rat 2, 3, 4. In WEC, we can directly target desired areas of embryos using fine glass capillaries because embryos can be manipulated under the microscope. Therefore, rodent WEC is very useful technique when we want to study dynamic developmental processes of postimplanted mammalian embryos. Up to date, several types of WEC systems have been developed 1. Among those, the rotator-type bottle culture system is most popular and suitable for long-term culture of embryos at midgestation, i.e., after E9.5 and E11.5 in the mouse and rat, respectively 1. In this video protocol, we demonstrate our standard procedures of rat WEC after E12.5 using a refined model of the original rotator system, which was designed by New and Cockroft 5, 6, and introduce various applications of WEC technique for studies in mammalian developmental biology.

Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development ...

Closed System Cell Culture Protocol Using HYPERStack Vessels with Gas Permeable Material Technology

Large volume adherent cell culture is currently standardized on stacked plate cell growth products when microcarrier beads are not an optimal choice. HYPERStack vessels allow closed system scale up from the current stacked plate products and delivers >2.5X more cells in the same volumetric footprint. The HYPERStack vessels function via gas permeable material which allows gas exchange to occur, therefore eliminating the need for internal headspace within a vessel. The elimination of headspace allows the compartment where cell growth occurs to be minimized to reduce space, allowing more layers of cell growth surface area within the same volumetric footprint.
For many applications such as cell therapy or vaccine production, a closed system is required for cell growth and harvesting. The HYPERStack vessel allows cell and reagent addition and removal via tubing from media bags or other methods.
This protocol will explain the technology behind the gas permeable material used in the HYPERStack vessels, gas diffusion results to meet the metabolic needs of cells, closed system cell growth protocols, and various harvesting methods.

An Introduction into the technology, protocol and handling of the Corning HYPERStack Vessels and accessories used for high yield adherent cell culture. The protocol will show how to use the closed system vessels for increasing cell harvesting over current stacked plate products.

Large volume adherent cell culture is currently standardized on stacked plate cell growth products when microcarrier beads are not an optimal choice.   ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

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 ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

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

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Culture and Maintenance of Human Embryonic Stem Cells

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

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Closed System Cell Culture Protocol Using HYPERStack Vessels with Gas Permeable Material Technology

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

A System for ex vivo Culturing of Embryonic Pancreas

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

An Ex vivo Culture System to Study Thyroid Development

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development