We thank Mr. Hajime Ichijo for video recording and helpful advice concerning editing the video. We also thank the Osumi lab members for animal care. This work is supported by a Grant-in-Aid for Scientific Research on Innovative Area, Neural Diversity and Neocortical Organization, from MEXT of Japan (to N. O.). T. K. was supported by a Research Fellowship of JSPS for Young Scientists.

NOTE: Animal experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The Committee for Animal Experimentation of the Tohoku University School of Medicine approved the experimental procedures described herein.

1. Anesthesia and Laparotomy
<ol style="margin-left: 40px;">
	<li>For blood collection, use specific pathogen-free male Sprague-Dawley rats. Fast the rats for at least 18 hr while providing water. Use retired male breeder rats at 6-8 months of age (550-650 g) for collecting blood.</li>
	<li>Flow 2.5-4.0% isoflurane, which is an inhalational anesthetic, into a box connected to an anesthesia apparatus to introduce anesthesia at 2.0-3.5 L/min for 10 min. Transfer rats into the box to introduce anesthesia.
	<ol>
		<li>Confirm anesthetization by checking for the loss of the response to stimulation on the paws with forceps. Cover the nose of the rats with a mask connected to an anesthesia apparatus to deeply anesthetize the rats during blood collection. The concentration and flow are maintained at 2.5-4.0% and 2.0-3.5 L/min, respectively. 
		NOTE: Halothane should not be used for inhalation anesthesia because embryos cultured in the serum that is collected from rats anesthetized with this reagent exhibit a delay in embryonic development compared to embryos cultured in the serum obtained from rats anesthetized with isoflurane22.</li>
	</ol>
	</li>
	<li>To avoid contamination of the fur, disinfect the skin by pouring 70% ethanol onto the abdomen of the anesthetized rat. Pick up the disinfected skin and abdominal wall at the lower region with a pair of forceps and cut these layers simultaneously toward the thorax region with a pair of large scissors to expose the internal organs. Reflect the gut outside of the abdominal cavity with a pair of forceps.</li>
	<li>Cut the skin and abdominal wall toward the hind limbs with a pair of large scissors to further expose the posterior abdominal region. Reflect the extra fat outside of the abdominal cavity with a pair of forceps.</li>
	<li>Pick up the visceral fat at the midline using two pairs of forceps and split the fat carefully in a parallel direction to expose the abdominal aorta. Repeat this procedure to split the visceral fat in a longitudinal direction to easily identify the bifurcation of the abdominal aorta. In addition, the aorta is pulsatile and can be distinguished from the adjacent vena cava using this characteristic.</li>
	<li>Pick up the fat around the abdominal aorta and the bifurcation carefully with two pairs of forceps and separate the fat on the abdominal aorta.</li>
</ol>
2. Blood Collection
<ol style="margin-left: 40px;">
	<li>Carefully insert the tip of a 21 G needle connected to a 20 ml syringe immediately cranial to the bifurcation of the aorta, with the bevel in a downward direction. Hold the syringe with one hand, and pull the syringe slowly to collect 15 ml of blood from a male rat.</li>
	<li>Remove the needle from the syringe, and pour the blood into two ice-cold 10-ml sterile test tubes (Figure 1A). Keep the tubes on ice until centrifugation to delay the formation of the blood clot. Reduce the centrifugation time by centrifuging several tubes at one time.</li>
	<li>Euthanize the anesthetized rat by thoracotomy and cutting the heart or decapitation.</li>
</ol>
3. Rat Serum Preparation
<ol style="margin-left: 40px;">
	<li>Centrifuge the collected blood for 5 min at 1,200 x g and room temperature (RT) to separate the blood into upper and lower layers (Figure 1C).</li>
	<li>Keep the tubes at 4 &#176;C for 1.5-2 hr to fix the fibrin clot.</li>
	<li>Pick up the fibrin clot in the upper layer using a pair of curved forceps, and squeeze the fibrin clot to separate the serum. Then, press the fibrin clot with the curved forceps facing downwards near the interface between the two layers (Figure 1D).</li>
	<li>Repeat step 3.3 to further detach the fibrin clot.</li>
	<li>Centrifuge the test tubes for 5 min at 1,200 x g and RT (Figure 1E).</li>
	<li>Carefully transfer the serum into a 15 ml sterile tube using a sterile pipette in a laminar air-flow cabinet.</li>
	<li>Centrifuge the collected rat serum for 5 min at 1,200 x g and RT to remove any remaining red cells.</li>
	<li>Pool the serum carefully into a new 15 ml sterilized tube using a sterile pipette to avoid contamination by residual red cells at the bottom of the tube (Figure 1F).</li>
	<li>Incubate the serum in a water bath at 56 &#176;C for 30 min to inactivate the complement system.</li>
	<li>Cool the tubes of rat serum to RT in the laminar air-flow cabinet. The serum should be aliquoted to 10 ml in individual sterile tubes. Store the tubes at -20 &#176;C until use (Figure 1H).</li>
</ol>

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No conflicts of interest are declared.

Reproducible results in WEC experiments are dependent on the use of high quality IC rat serum and accurate embryo dissection technique3. Do not shorten the period of fasting before collecting blood because fasting is necessary to standardize glucose concentrations in the blood. Female rats should not be used for the preparation of serum because hormone levels change in female animals according to the estrous cycle, and such changes in estradiol hormones are unsuitable for embryo cultures. In addition, extremel...

A variety of model animals are used in developmental biology to investigate developmental mechanisms at the molecular and cellular levels. For example, amphibian and avian species have been widely used as classical model animals that are suitable for direct manipulation of embryos because these embryos develop outside of the mother. In contrast to these animals, mammalian embryos grow in the uterus of the mother, and growth at later stages is crucially dependent on the function of the uterus. Therefore, it is typically difficult to directly manipulate mammalian embryos such as those from the mouse and rat at early stages. In the 1960s, Denis New established a mammalian whole embryo culture (WEC) technique using WEC apparatuses with a continuous oxygen supply and heat control1. In WEC, mouse and rat embryos can grow ex vivo, (i.e., outside of the uterus). Although the WEC technique was often used in teratology by adding various chemical compounds into the culture medium, this technique has also been used in various developmental biology studies to examine unique developmental mechanisms in mammals2-4. For example, WEC is combined with other techniques, such as cell labeling, in wild-type and mutant embryos by using fluorescent dye5, cell transplantation6, and gene introduction via lipofection7 and electroporation8-13.
Recently, in utero manipulation has been used to analyze developmental processes in rodent embryos at later stages and has been combined with electroporation techniques14-16. However, these techniques are not suitable for the manipulation of postimplantation and midgestation embryos due to difficulties achieving accurate local injection of the DNA solution into embryos at the early stages. Although ultrasound-guided cell transplantation and injection of viral vectors into early embryos (i.e., E8.5-E-9.5 in the mouse) in utero have been reported previously17,18, excellent skills are required to perform these experiments with a high success rate. Therefore, WEC with high accessibility ex vivo has advantages with respect to the manipulation of mouse and rat embryos.
Immediately centrifuged (IC) rat serum prepared from male rats is often used for WEC medium. When embryos are cultured at the postimplantation stage (i.e., earlier than E8.0 in the mouse or E10.0 in the rat), a mixture of synthetic medium and IC rat serum is often used as a medium for WEC19. However, to culture embryos at mid-gestation (i.e., E8.0-12.5 in mouse embryos or E10.0-E14.5 in rat embryos), 100% serum should be used as a medium because no currently available alternative media allows embryos to grow normally in vitro for more than 2 days.
The preparation of high-quality rat serum is a critical step for achieving reproducibility in WEC experiments. Compared to delayed centrifugation, IC has the benefit of reducing hemolysis when the serum is collected by squeezing the fibrin clot because most of the red blood cells have already been separated from the fibrin clot. As hemolytic rat serum fails to support the normal growth of rat and mouse embryos, the preparation of serum using immediate centrifugation is preferable to preparation using delayed centrifugation. Our protocol contains two additional steps compared to other protocols18-20 (i.e., storing the collected blood on ice prior to the first centrifugation and maintaining the collected blood samples for 2 hr at 4 &#176;C after the first centrifugation). The former step can delay the formation of the blood clot, and the latter step promotes the solidification of the fibrin clot for easy squeezing. Therefore, our protocol can be used by beginners. However, reproducing blood collection and serum extraction accurately by simply referring to protocol books is extremely difficult19-21. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, which includes blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

<table border="0" cellpadding="0" cellspacing="0" width="892">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:156px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:401px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Isoflurane</td>
			<td>Abbott</td>
			<td>B506</td>
			<td>For anesthesia of rats.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Large scissors</td>
			<td>Napox</td>
			<td>B-7H</td>
			<td>Stelized scissors for cutting the skin and muscle of rats.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Curved forceps</td>
			<td>Napox</td>
			<td>A-3-2</td>
			<td>Stelized forceps for picking up the skin of rats and squeezing the fibrin clot.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sprague-Dawley (SD) rat</td>
			<td>Charles Rivers Laboratories</td>
			<td></td>
			<td>Retired male rats from colony in the lab or purchased retired male rats.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe (20 ml)</td>
			<td>TERUMO</td>
			<td>SS-29ESZ</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Needle (21G x 5/8&#34;)</td>
			<td>TERUMO</td>
			<td>NN-2116R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile test (spitz) tube (10 ml)</td>
			<td>ASIAKIZAI</td>
			<td style="width:119px;">1101C000B-10</td>
			<td>For collection of boold</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile disposable pipette</td>
			<td>Eiken Chemical</td>
			<td>CD2000 No.4</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterilie disposable tube (15 ml)&#160;</td>
			<td>Falcon</td>
			<td>2196</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Figure 1 shows representative results of the described procedures for the separation of blood cells and serum. We typically obtain 15 ml of blood from a retired male rat (Figure 1A). By centrifugation, the collected blood can be separated into an upper layer containing the serum and the fibrin clot, which is indicated with an arrow, and a lower layer containing the blood cells (Figure 1C). Importantly, hemolytic blood samples cannot be separated into serum and blood laye...

Watch this Scientific Journal Video about Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture at JoVE.com

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

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 ...

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13.7K Views.  Jichi Medical University. 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.