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><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 (10,000 IU/ml); Streptomycin (10,000 &amp;micro;g/ml; Amphotericin (250 &amp;micro;g/ml)&amp;nbsp;</td></tr><tr><td>DMEM&amp;nbsp;&amp;nbsp;</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% O&lt;sub&gt;2&lt;/sub&gt;/5% CO&lt;sub&gt;2&lt;/sub&gt; 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>CO&lt;sub&gt;2&lt;/sub&gt; 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</td><td>AG285</td><td></td></tr><tr><td>Centrifuge Tube, 50 ml</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&amp;nbsp;</td><td>Roboz</td><td>RS-6812</td><td></td></tr><tr><td>Microdissecting Forceps, 4 in</td><td>Roboz</td><td>RS-5211</td><td></td></tr><tr><td>Microdissecting Forceps - Hudson (cWALD), 4-3/4 in</td><td>Roboz</td><td>RS-5237</td><td></td></tr><tr><td>Microdissecting Tweezers (5/45)</td><td>Roboz</td><td>RS-5005</td><td>Modified - Sharp ends were made blunt</td></tr><tr><td>Microdissecting Tweezers (5)</td><td>Roboz</td><td>RS-5060</td><td>Modified - Sharp ends were made blunt</td></tr><tr><td>Microdissecting 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-100 mm</td><td>Fisher Scientific</td><td>087571Z</td><td></td></tr><tr><td>Petri Dish-60 mm</td><td>Fisher Scientific</td><td>0875713A</td><td></td></tr><tr><td>Petri Dish-35 mm</td><td>Fisher Scientific</td><td>0875711YZ</td><td></td></tr><tr><td>Filter System ( 0.22 &amp;micro;m Cellulose Acetate)-150 ml</td><td>Corning</td><td>431153</td><td></td></tr><tr><td>Filter System (0.22 &amp;micro;m Cellulose Acetate)-250 ml</td><td>Corning</td><td>430756</td><td></td></tr><tr><td>Filter System (0.22 &amp;micro;m Cellulose Acetate)-500 ml</td><td>Corning</td><td>430758</td><td></td></tr><tr><td>Pipet-aid Pipettor</td><td>Drummond Scientific Co.</td><td>D57849</td><td></td></tr><tr><td>Serological Pipette-10 ml</td><td>VWR</td><td>89130-898</td><td></td></tr><tr><td>Disposable Serological Pipette-25 ml</td><td>Corning</td><td>4251</td><td></td></tr><tr><td>Transfer Pipette - 7.7 ml</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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Research

JoVE Journal

Biology

25.9K Views.  University of Georgia.  Serum gebruikt in embryocultures bevat onbekende componenten die het resultaat van experimenten vooral in studies met signalering interacties beïnvloeden. Hier gebruikte we een serumvrij zuurstof kweeksysteem aantonen dat mid-dracht muizenembryo's gekweekt gedurende 16-40 uur vertonen morfologische ontwikkeling vergelijkbaar embryo ontwikkeling in utero. 

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

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

Developmental Biology

Neural Tube Closure in Mouse Whole Embryo Culture

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray &amp; Ross, 2009; Ross, 2010). However, the study of mouse embryos in utero is limited by our inability to directly pharmacologically manipulate the embryos in isolation from the effects of maternal metabolism on the reagent of interest. Whether using a small molecule, recombinant protein, or siRNA, delivery of these substances to the mother, through the diet or by injection will subject these unstable compounds to a variety of bodily defenses that could prevent them from reaching the embryo. Investigations in cultures of whole embryos can be used to separate maternal from intrinsic fetal effects on development.
Here, we present a method for culturing mouse embryos using highly enriched media in a roller incubator apparatus that allows for normal neural tube closure after dissection (Crockett, 1990). Once in culture, embryos can be manipulated using conventional in vitro techniques that would not otherwise be possible if the embryos were still in utero. Embryo siblings can be collected at various time points to study different aspects of neurulation, occurring from E7-7.5 (neural plate formation, just prior to the initiation of neurulation) to E9.5-10 (at the conclusion of cranial fold and caudal neuropore closure, Kaufman, 1992). In this protocol, we demonstrate our method for dissecting embryos at timepoints that are optimal for the study of cranial neurulation. Embryos will be dissected at E8.5 (approx. 10-12 somities), after the initiation of neural tube closure but prior to embryo turning and cranial neural fold closure, and maintained in culture till E10 (26-28 somities), when cranial neurulation should be complete.

A method allowing for direct pharmacological manipulation of mouse embryos during neurulation that bypasses maternal metabolism is described. The technique can be adapted to study different aspects of neurulation by varying the time point and pharmacological agent.

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray &amp; Ross, 2009; Ross, 2010).  However, the study of mouse ...

Neuroscience

Collection of Serum- and Feeder-free Mouse Embryonic Stem Cell-conditioned Medium for a Cell-free Approach

The capacity of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to generate various cell types has opened new avenues in the field of regenerative medicine. However, despite their benefits, the tumorigenic potential of ESCs and iPSCs has long been a barrier for clinical applications. Interestingly, it has been shown that ESCs produce several soluble factors that can promote tissue regeneration and delay cellular aging, suggesting that ESCs and iPSCs can also be utilized as a cell-free intervention method. Therefore, the method for harvesting mouse embryonic stem cell (mESC)-conditioned medium (mESC-CM) with minimal contamination of serum components (fetal bovine serum, FBS) and feeder cells (mouse embryonic fibroblasts, MEFs) has been highly demanded. Here, the present study demonstrates an optimized method for the collection of mESC-CM under serum- and feeder-free conditions and for the characterization of mESC-CM using senescence-associated multiple readouts. This protocol will provide a method to collect pure mESC-specific secretory factors without serum and feeder contamination.

This protocol provides a method for the collection of mouse embryonic stem cell (mESC)-conditioned medium (mESC-CM) derived from serum (fetal bovine serum, FBS)- and feeder (mouse embryonic fibroblasts, MEFs)-free conditions for a cell-free approach. It may be applicable for the treatment of aging and aging-associated diseases.

The capacity of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to generate various cell types has opened new avenues in the field ...

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a general challenge in developmental biology research. In the embryonic heart, this can be particularly difficult as regional differences in the expression of transcriptional regulators, paracrine/juxtacrine signaling cues, and hemodynamic force are all known to influence cardiomyocyte maturation. A simplified method to alter a developing cardiomyocyte's molecular and biomechanical microenvironment would, therefore, serve as a powerful technique to examine how local conditions influence cell fate and function. To address this, we have optimized a method to physically transplant juvenile cardiomyocytes into ectopic locations in the heart or the surrounding embryonic tissue. This allows us to examine how microenvironmental conditions influence cardiomyocyte fate transitions at single cell resolution within the intact embryo. Here, we describe a protocol in which embryonic myocytes can be isolated from a variety of cardiac sub-domains, dissociated, fluorescently labeled, and microinjected into host embryos with high precision. Cells can then be directly analyzed in situ using a variety of imaging and histological techniques. This protocol is a powerful alternative to traditional grafting experiments that can be prohibitively difficult in a moving tissue such as the heart. The general outline of this method can also be adapted to a variety of donor tissues and host environments, and its ease of use, low cost, and speed make it a potentially useful application for a variety of developmental studies.

In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a ...

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 ...

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. ...

Live Imaging of Early Cardiac Progenitors in the Mouse Embryo

The first steps of heart development imply drastic changes in cell behavior and differentiation. While analysis of fixed embryos allows studying in detail specific developmental stages in a still snapshot, live imaging captures dynamic morphogenetic events, such as cell migration, shape changes, and differentiation, by imaging the embryo as it develops. This complements fixed analysis and expands the understanding of how organs develop during embryogenesis. Despite its advantages, live imaging is rarely used in mouse models because of its technical challenges. Early mouse embryos are sensitive when cultured ex vivo and require efficient handling. To facilitate a broader use of live imaging in mouse developmental research, this paper presents a detailed protocol for two-photon live microscopy that allows long-term acquisition in mouse embryos. In addition to the protocol, tips are provided on embryo handling and culture optimization. This will help understand key events in early mouse organogenesis, enhancing the understanding of cardiovascular progenitor biology.

We present a detailed protocol for mouse embryo culture and imaging that enables 3D + time imaging of cardiac progenitor cells. This video-toolkit addresses the key skills required for successful live imaging otherwise hard to acquire from text-only publications.

The first steps of heart development imply drastic changes in cell behavior and differentiation. While analysis of fixed embryos allows studying in detail ...

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for ...

Evaluating Embryo Adhesion in an In Vitro Co-Culture Model with Ishikawa Cells

Establishment of an Embryo Implantation Model In Vitro

Embryo implantation is the first step in the establishment of a successful pregnancy. An in vitro model for embryo implantation is critical for basic biological research, drug development, and screening. This paper presents a simple, rapid, and highly efficient in vitro model for embryo implantation. In this protocol, we first introduce mouse blastocyst acquisition and human endometrial adenocarcinoma cells (Ishikawa)&#160;preparation for implantation, followed by the co-culture method for mouse embryos and Ishikawa cells. Finally, we conducted a study to assess the impact of varying concentrations of 17-&#946;-estradiol (E2) and progesterone (P4) on embryo adhesion rates based on this model. Our findings revealed that high concentrations of E2 significantly reduced embryo adhesion, whereas the addition of progesterone could restore the adhesion rate. This model offers a simple and fast platform for evaluating and screening molecules involved in the adhesion process, such as cytokines, drugs, and transcription factors controlling implantation and endometrial receptivity.

Author Spotlight: Advancing Therapeutic Strategies for Improving Pregnancy Rates by Analyzing Embryo-Endometrium Interactions

This protocol describes methodologies to establish simple and efficient embryo implantation in vitro model for evaluating the relevant molecules affecting the embryo implantation process.

Embryo implantation is the first step in the establishment of a successful pregnancy. An in vitro model for embryo implantation is critical for basic ...

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

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