We thank Mr. Hajime Ichijo for video-recording and helpful advices for editing the video. We also thank Drs.Yuji Tsunekawa and Kaichi Yoshizaki for kind assistant for video-recording. This work is supported by KAKENHI on Young Scientist B and on Priority Areas- Molecular Brain Science from MEXT of Japan. We acknowledge the support of Global COE Program "Basic and Translational Research Center for Global Brain Science" from MEXT of Japan and The Core Research for Evolutional Science and Technology (CREST) from Japanese Science and Technology Corporation from Japanese Science and Technology Agency (JST).

1. Setting up the WEC system
<ol>
	<li>Joint a 0.22 &mu;m membrane filter to the inlet silicon tube within WEC system a).</li>
	<li>Open the valve of a gas cylinder containing O2 (95%) and CO2 (5%). Flow the gas mixture into the drum via the bubbling bottle containing autoclaved water.</li>
	<li>Adjust the flow volume of gas mixture to 50 cc.</li>
	<li>Insert the out let silicon tube into a water bottle and check out the flow of gas mixture.</li>
	<li>Rotate the drum at the speed of 20 rpm/min.</li>
</ol>
2. Preparation of culture medium
<ol>
	<li>Thaw rat immediately-centrifuged (IC) serum at 37.0&deg;C b). Add D-glucose (2 mg/ml) into thawed serum in an autoclaved 100 ml beaker. Culture medium should be prepared at the primary culture level (e.g., using a hood).</li>
	<li>Add antibiotic-antimycotic (100X) liquid to the serum with 1: 400 dilution.</li>
	<li>Vaporize remaining isoflurane (due to anesthesia of rats during collecting blood) from the serum for 20 minutes at room temperature (RT) c).</li>
	<li>Sterilize the culture medium using a 0.45 &mu;m membrane filter.</li>
	<li>Pour 3.0 ml of the medium in each culture bottle and seal the top of each bottle with a silicon plug, covered with aluminum foil (see ref. 14) d).</li>
	<li>Prepare three or four disposal Petri dishes containing Tyrode's saline e).</li>
</ol>
3. Anesthesia of the rat and isolation of the uterus
<ol>
	<li>Anesthetize a timed-pregnant rat deeply with inhalational or parenteral anesthetics.</li>
	<li>Clean the abdominal area of the anesthetized pregnant rat with 70% ethanol.</li>
	<li>Pick up the skin with forceps and exteriorize the abdominal wall by cutting the skin with large scissors f).</li>
	<li>Pick up the abdominal wall with a pair of forceps and make U-shaped large longitudinal incisions on the abdominal wall with large scissors up to the thoracic level g).</li>
	<li>Bear away the intestine to the left side with a pair of forceps and expose one end of the uterus connected to the ovary.</li>
	<li>Pick up the end of the uterus with a pair of forceps and cut the position between the uterus and ovary with large scissors.</li>
	<li>Take out the uterus from the pregnant rat by cutting the fat around the uterus until the other end of the uterus.</li>
	<li>Transfer the uterus to a Petri dish containing Tyrode's saline.</li>
	<li>Euthanize the rat by excessive administration of anesthetics or cervical dislocation after removing the uterus.</li>
</ol>
4. Removal of embryos from the uterus
<ol>
	<li>Briefly rinse the uterus in Tyrode's saline and transfer it to the second Petri dish with a pair of fine forceps h).</li>
	<li>Under a binocular microscope, pick up the uterine wall with a pair of fine forceps and cut the uterine wall at the side opposite to the mesometrium connecting with blood vessels using ophthalmic straight scissors.</li>
	<li>Insert the tip of ophthalmic straight scissors into the space between the uterine wall and decidua. Cut the uterine wall longitudinally along the antimesometrial side.</li>
	<li>Clump the decidua at the mesometrial side and separate conceptuses from the uterus using two pairs of fine forceps i).</li>
	<li>Transfer conceptuses to the third Petri dish with a sterilized glass pipette with a suitable diameter.</li>
</ol>
5. Dissection of rat embryos
<ol>
	<li>Insert one tip of the microforceps into the decidua and make a transverse incision on the decidua around the conceptus with two pairs of forceps.</li>
	<li>Insert the tips of the forceps into the decidua at the placental side and make two longitudinal incisions on the decidua to the top.</li>
	<li>Remove the decidua at the placental side by ripping out that with two pairs of forceps. Remove the remaining decidua with two pairs of forceps.</li>
	<li>Pick up Reichert's membrane, make a horizontal incision on it, and separate the membrane from the conceptus at the anti-placental side.</li>
	<li>Make a small hole on the yolk sac near the head of the embryo with the two pairs of forceps, and cut the yolk sac with a pair of ophthalmic curved scissors. It is important not to damage major blood vessels.</li>
	<li>Pick up the amnion at the site around the head of the embryo with the two pairs of fine forceps, and gently pull out the embryo from the yolk sac by tearing the amnion h). The body of the embryo should be pulled out of the yolk sac to increase the supply of oxygen for the embryo at the midgestation stage.</li>
</ol>
6. Whole embryo culture
<ol>
	<li>Check damages on the placenta, yolk sac, and the condition of heart beating and blood circulation.</li>
	<li>Being aware of not stretching the umbilical cord, transfer the embryo to a culture bottle with a sterilized glass pipette. Carry over of Tyrode's saline to culture bottles should be as little as possible.</li>
	<li>Remove a non-hole silicon plug from the rotator drum. Connect the culture bottle with a plug with a hole to the rotator drum of the WEC system perpendicularly. Be careful not to put force on the drum in unsuitable directions since the rotator drum is a very fine equipment. The bottles containing the dissected embryo are transferred to culture one by one; opening of the front door of the incubator should be minimized to avoid the temperature of the incubator is decreased j).</li>
	<li>After 10 hr of culture, increase the flow volume of gas mixture up to 75 cc and up to 100 cc after 24 hr (Table 1).</li>
	<li>The culture medium should be changed around 24 hr by transferring the cultured embryo into a new culture bottle with freshly prepared medium. Increase the flow volume up to 125 cc after 34 hr and up to 150 cc after 48 hr (Table 1)</li>
	<li>Occasionally check the condition of the cultured embryos by counting heartbeat rate (i.e., 120-150/min is optimal) and by observing blood circulation. If required count the number of somites to judge the growth of embryos in Tyrode's saline. In normal development, one somite is added in two hours.</li>
	<li>When WEC is finished, stop supply of gas mixture and disconnect the inlet tube and the membrane filter to avoid back flow into the inlet tube from the bubbling bottle.</li>
</ol>
7. Notes
<ol class="lalpha">
	<li>The temperature inside the WEC system is constantly kept at 37.0-37.5&deg;C.</li>
	<li>100% rat IC serum is routinely used in our WEC for rat and mouse embryos, and kept at -20&deg;C before use.</li>
	<li>We purchase rat IC serum, which is collected from rats that are anesthetized with isoflurane.</li>
	<li>Disposable bottles are also available from Ikemoto Rika. Bottles and silicon plugs should be autoclaved.</li>
	<li>Dissection of embryos should be performed at RT because maintaining of embryos at low or high temperature during dissection affect subsequent development in WEC.</li>
	<li>Tools for dissection are sterilized by dry heat.</li>
	<li>The skin and abdominal wall should be cut separately to avoid contamination of hairs into a dish as possible. We change a pair of large scissors and forceps with a new pair of them when we cut the skin and isolate the uterus, respectively.</li>
	<li>Using forceps whose tips are fine is very important to dissect tissues certainly. We manually sharpen the tips of forceps with a stone and machine oil.</li>
	<li>We use one side of the forceps like a knife to avoid excess pressure to embryos.</li>
	<li>When the temperature inside the WEC system decreases by opening the door repeatedly, we continue to turn on the light to keep temperature inside the system.</li>
</ol>
8. Representative results
Figure 1 shows procedures of dissection of the rat embryo and cultured rat embryos.
<img src="/files/ftp_upload/2170/2170fig1.jpg" alt="Figure 1" /> Figure 1. Whole embryo culture of the E12.5 rat embryo. (A) A conceptus dissected from the uterus of the pregnant rat. (B) Removal of the decidua at the placenta side. (C) Removal of Reichert's membrane. (D) Opening the yolk sac. (E) The rat embryo culturing in the bottle for 6 hr after the beginning of WEC. (F) The rat embryo cultured for 42 hr. d, decidua; R; Reichert's membrane; p, placenta; ys, yolk sac; e, embryo.
<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>0 hr</td>
			<td>12 hr</td>
			<td>24 hr</td>
			<td>36 hr</td>
			<td>48 hr</td>
		</tr>
		<tr>
			<td>E12.5 rat embryo</td>
			<td>95% 50 cc</td>
			<td>95% 75 cc (10 hr)</td>
			<td>95% 100 cc</td>
			<td>95% 125 cc (34 hr)</td>
			<td>95% 150 cc</td>
		</tr>
	</tbody>
</table>
Table 1. Optimal oxygen condition.

<ol>
	<li>New, D. A. T., Copp, A. J., Cockroft, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intoroduction.+In+mammalian+postimplantation+embryos.">Intoroduction. In mammalian postimplantation embryos.</a> In Mammalian Postimplantation Embryos A Practical Approach. , 1-14 (1990).</li><li>Cockroft, D. L., Copp, A. J., Cockroft, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+and+culture+of+post+implantation+embryos.">Dissection and culture of post implantation embryos.</a> In Mammalian Postimplantation Embryos A Practical Approach. , 15-40 (1990).</li><li>Morris-Kay, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postimplantation+mammalian+embryos.">Postimplantation mammalian embryos.</a> Essential Developmental Biology A Practical Approach. , 55-66 (1993).</li><li>Fujinaga, M., Tuan, R. S., Lo, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+culture+of+rodent+embryos+during+the+early+postimplantation+period.">In vitro culture of rodent embryos during the early postimplantation period.</a> Developmental Biology Protocols. , 53-76 (2000).</li><li>Eto, K., Takakubo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+development+of+rat+embryos+in+culture+during+the+period+of+craniofacial+morphogenesis.">Improved development of rat embryos in culture during the period of craniofacial morphogenesis.</a> J. Craniofac. Genet. Dev. Biol. 5, 351-355 (1985).</li><li>New, D. A., Cockroft, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rotating+bottle+culture+method+with+continuous+replacement+of+the+gas+phase.">A rotating bottle culture method with continuous replacement of the gas phase.</a> Experientia. 35, 138-140 (1979).</li><li>Osumi-Yamashita, N., Ninomiya, Y., Eto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+craniofacial+embryology+in+vitro.">Mammalian craniofacial embryology in vitro.</a> Int. J. Dev. Biol. 41, 187-194 (1997).</li><li>Inoue, T., Nakamura, S., Osumi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+mapping+of+the+mouse+prosencephalic+neural+plate.">Fate mapping of the mouse prosencephalic neural plate.</a> Dev. Biol. 219, 373-383 (2000).</li><li>Nomura, T., Holmberg, J., Frisen, J., Osumi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pax6-dependent+boundary+defines+alignment+of+migrating+olfactory+cortex+neurons+via+the+repulsive+activity+of+ephrin+A5.">Pax6-dependent boundary defines alignment of migrating olfactory cortex neurons via the repulsive activity of ephrin A5.</a> Development. 133, 1335-1345 (2006).</li><li>Inoue, Y. U., Asami, J., Inoue, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+labeling+of+mouse+rhombomeres+by+Cadherin-6::EGFP-BAC+transgenesis+underscores+the+role+of+cadherins+in+hindbrain+compartmentalization.">Genetic labeling of mouse rhombomeres by Cadherin-6::EGFP-BAC transgenesis underscores the role of cadherins in hindbrain compartmentalization.</a> Neurosci. Res. 63, 2-9 (2009).</li><li>Calegari, F., Huttner, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+inhibition+of+cyclin-dependent+kinases+that+lengthens,+but+does+not+arrest,+neuroepithelial+cell+cycle+induces+premature+neurogenesis.">An inhibition of cyclin-dependent kinases that lengthens, but does not arrest, neuroepithelial cell cycle induces premature neurogenesis.</a> J. Cell Sci. 116, 4947-4955 (2003).</li><li>Osumi, N., Inoue, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+into+cultured+mammalian+embryos+by+electroporation.">Gene transfer into cultured mammalian embryos by electroporation.</a> Methods. 24, 35-42 (2001).</li><li>Arai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Fabp7,+a+downstream+gene+of+Pax6,+in+the+maintenance+of+neuroepithelial+cells+during+early+embryonic+development+of+the+rat+cortex.">Role of Fabp7, a downstream gene of Pax6, in the maintenance of neuroepithelial cells during early embryonic development of the rat cortex.</a> J. Neurosci. 25, 9752-9761 (2005).</li><li>Takahashi, M., Nomura, T., Osumi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transferring+genes+into+cultured+mammalian+embryos+by+electroporation.">Transferring genes into cultured mammalian embryos by electroporation.</a> Dev. Growth Differ. 50, 485-497 (2008).</li></ol>

No conflicts of interest declared.

There are two critical steps in rodent WEC for the success. First, the dissection procedure should be accurate not to damage embryos, especially blood vessels. Second, the procedure should be as quickly as possible because oxygen and nutrient are no longer supplied via the placenta after isolation from the uterus. This is critical for older embryos. In the case of rat WEC after E12.5, we should transfer dissected embryos into culture bottles within 30 minutes.
We have analyzed migration patte...

<table border="1">
 <tbody>
 <tr>
 <td>Name</td>
 <td>Type</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comment</td>
 </tr>
 <tr>
 <td colspan="5">Setting up WEC </td>
 </tr>
 <tr>
 <td>WEC system (10-0310)</td>
 <td>Tool</td>
 <td>Ikemoto Rika</td>
 <td>010-0310</td>
 <td>Another small model is also available.</td>
 </tr>
 <tr>
 <td>Silicon plug without a hole</td>
 <td>Tool</td>
 <td>Ikemoto Rika</td>
 <td>010-032-08</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Gas mixture cylinder</td>
 <td>Tool</td>
 <td>Nikko Sanso</td>
 <td>-</td>
 <td>Containing 95% oxygen &amp; 5% carbon dioxide. Custom order.</td>
 </tr>
 <tr>
 <td>Gas regulator</td>
 <td>Tool</td>
 <td>Ono Seisakusho</td>
 <td>WR-11</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.22 &mu;m filter Millex GS</td>
 <td>Tool</td>
 <td>Millipore</td>
 <td>SLGS033SS</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td colspan="5">Anesthesia and isolation of the uterus </td>
 </tr>
 <tr>
 <td>Large scissors</td>
 <td>Tool</td>
 <td>Napox</td>
 <td>B-7H</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Forceps</td>
 <td>Tool</td>
 <td>Napox</td>
 <td>A-3-2</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Disposal Petri dish 
 (90 mm x 15 mm) </td>
 <td>Tool</td>
 <td>Iwaki</td>
 <td>SH90-15</td>
 <td>Deep-type dish is the best for dissection.</td>
 </tr>
 <tr>
 <td>Isoflurane</td>
 <td>Reagent</td>
 <td>Abbott</td>
 <td>B506</td>
 <td>For anesthesia</td>
 </tr>
 <tr>
 <td>Pentobarbital sodium</td>
 <td>Reagent</td>
 <td>Schering-Plough Animal Health</td>
 <td>-</td>
 <td>For anesthesia</td>
 </tr>
 <tr>
 <td>Tyrode's saline</td>
 <td>Reagent</td>
 <td>-</td>
 <td>-</td>
 <td>According as the protocol in Ref. 2. Save at 4&deg;C. </td>
 </tr>
 <tr>
 <td>Timed-pregnant Sprague-Dawley rat</td>
 <td>Animal</td>
 <td>Charles Rivers Laboratories Japan</td>
 <td>-</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td colspan="5">Preparation of culture medium </td>
 </tr>
 <tr>
 <td>Culture glass bottle</td>
 <td>Tool</td>
 <td>Ikemoto Rika</td>
 <td>010-032-05</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Disposal culture bottle</td>
 <td>Tool</td>
 <td>Ikemoto Rika</td>
 <td>010-032-06</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Silicon plug with hole</td>
 <td>Tool</td>
 <td>Ikemoto Rika</td>
 <td>010-032-07</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Aluminum foil</td>
 <td>Tool</td>
 <td>Any company</td>
 <td>-</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Autoclaving bag</td>
 <td>Tool</td>
 <td>Hogy</td>
 <td>HM-26</td>
 <td>For culture bottles</td>
 </tr>
 <tr>
 <td>Autoclaving bag</td>
 <td>Tool</td>
 <td>Hogy</td>
 <td>HM-14A</td>
 <td>For silicon plugs</td>
 </tr>
 <tr>
 <td>0.45 &mu;m filter Millex HA</td>
 <td>Tool</td>
 <td>Millipore</td>
 <td>SLHA033SS</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>50 ml conical tube</td>
 <td>Tool</td>
 <td>Becton Dickinson</td>
 <td>352070</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>100 ml beaker</td>
 <td>Tool</td>
 <td>Iwaki</td>
 <td>TE-32</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Rat IC serum</td>
 <td>Reagent</td>
 <td>Charles Rivers Laboratories Japan</td>
 <td>-</td>
 <td>Refer to: Mr. Kunihiko Morisaki, 
 TEL: +81-(0) 45-474-9336; FAX: +81-(0) 45-474-9340</td>
 </tr>
 <tr>
 <td>D(+)-Glucose</td>
 <td>Reagent</td>
 <td>Wako</td>
 <td>041-00595</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Antibiotic-antimyotic liquid</td>
 <td>Reagent</td>
 <td>Gibco</td>
 <td>15240</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td colspan="5">Dissection of embryos </td>
 </tr>
 <tr>
 <td>Ophthalmic straight scissors</td>
 <td>Tool</td>
 <td>Napox</td>
 <td>MB50-7</td>
 <td>For cutting the uterine wall</td>
 </tr>
 <tr>
 <td>Ophthalmic curved scissors</td>
 <td>Tool</td>
 <td>Napox</td>
 <td>MB54-2</td>
 <td>For cutting the yolk sac</td>
 </tr>
 <tr>
 <td>Forceps #5 </td>
 <td>Tool</td>
 <td>Vigor</td>
 <td>T6715</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Forceps #5F</td>
 <td>Tool</td>
 <td>Regine</td>
 <td>T6819</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glass pipette</td>
 <td>Tool</td>
 <td>-</td>
 <td>-</td>
 <td>Made by hand using Pasteur pipette.</td>
 </tr>
 <tr>
 <td>Autoclaving bag</td>
 <td>Tool</td>
 <td>Hogy</td>
 <td>HM-4</td>
 <td>For glass pipettes</td>
 </tr>
 <tr>
 <td>Binocular microscope</td>
 <td>Tool</td>
 <td>Leica</td>
 <td>Mz7s</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Light</td>
 <td>Tool</td>
 <td>Leica</td>
 <td>CLS 150XD</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Oil stone</td>
 <td>Tool</td>
 <td>Uchida Yoko</td>
 <td>833-2000</td>
 <td>For sharpening of forceps</td>
 </tr>
 <tr>
 <td>Machine oil</td>
 <td>Tool</td>
 <td>Uchida Yoko</td>
 <td>835-0000</td>
 <td>For sharpening of forceps</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System at JoVE.com

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

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 ...

the-method-rodent-whole-embryo-culture-using-rotator-type-bottle

Research

JoVE Journal

Biology

23.6K Views.  Tohoku University Graduate School of Medicine. Tutta la tecnica di coltura degli embrioni del mouse ci permette di cultura e di embrioni di ratto Ex vivo durante i periodi limitati corrispondenti a fasi midgestation. In questo protocollo video, abbiamo dimostrato le nostre procedure standard di coltura degli embrioni di ratto dopo tutto E12.5 con il rotore di tipo sistema cultura bottiglia.

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

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 ...

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 ...

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 ...

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

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

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

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

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental ...

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 ...

Establishment of an Extracellular Acidic pH Culture System

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported.
Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...

Evaluating the Angiogenetic Properties of Ovarian Cancer Stem-Like Cells using the Three-Dimensional Co-Culture System, NICO-1

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular matrix. The ability of CSCs to participate in the development of endothelium constitutes an important characteristic that directly contributes to the general understanding of the mechanisms of tumorigenesis and tumor metastasis. The purpose of this work is to establish a reproducible methodology to investigate the tumor-initiation capability of ovarian cancer stem cells (OCSCs). Herein, we examined the neovascularization mechanism between endothelial cells and OCSCs along with the morphological changes of endothelial cells using the in vitro co-culture model NICO-1. This protocol allows visualization of the neovascularization step surrounding the OCSCs in a time course manner. The technique can provide insight regarding the angiogenetic properties of OCSCs in tumor metastasis.

Ovarian cancer stem cells (OCSC) are responsible for cancer initiation, recurrence, therapeutic resistance, and metastasis. The OCSC vascular niche is considered to promote self-renewal of OCSCs, leading to chemoresistance. This protocol provides the basis for establishing a reproducible OCSC vascular niche model in vitro.

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular ...

Process Development for the Production and Purification of Adeno-Associated Virus (AAV)2 Vector using Baculovirus-Insect Cell Culture System

Adeno-associated viruses (AAV) are promising vectors for gene therapy applications. Here, the AAV2 vector is produced by co-culture of Spodoptera frugiperda (Sf9) cells with Sf9 cells infected with baculovirus (BV)-AAV2-GFP (or therapeutic gene) and BV-AAV2-rep-cap in serum-free suspension culture. Cells are cultured in a flask in an orbital shaker or Wave bioreactor. To release the AAV particles, producer cells are lysed in buffer containing detergent, which is subsequently clarified by low-speed centrifugation and filtration. AAV particles are purified from the cell lysate using AVB Sepharose column chromatography, which binds AAV particles. Bound particles are washed with PBS to remove contaminants and eluted from the resin using sodium citrate buffer at pH 3.0. The acidic eluate is neutralized with alkaline Tris-HCl buffer (pH 8.0), diluted with phosphate-buffered saline (PBS), and further concentrated with tangential flow filtration (TFF). The protocol describes small-scale pre-clinical vector production compatible with scale-up to large-scale clinical-grade AAV manufacturing for human gene therapy applications.

Process Development for the Production and Purification of Adeno-Associated Virus AAV2 Vector using Baculovirus-Insect Cell Culture System

In this protocol, AAV2 vector is produced by co-culturing Spodoptera frugiperda (Sf9) insect cells with baculovirus (BV)-AAV2-green fluorescent protein (GFP) or therapeutic gene and BV-AAV2-rep-cap infected Sf9 cells in suspension culture. AAV particles are released from the cells using detergent, clarified, purified by affinity column chromatography, and concentrated by tangential flow filtration.