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>

<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=Introduction.">Introduction.</a> Mammalian Postimplantation Embryos. A Practical Approach. , 1-14 (1990).</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>Takahashi, M., 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=The+method+of+rodent+whole+embryo+culture+using+the+rotator-type+bottle+culture+system.">The method of rodent whole embryo culture using the rotator-type bottle culture system.</a> J Vis Exp. (42), e2170 (2010).</li><li>New, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+embryo+culture,+teratogenesis,+and+the+estimation+of+teratologic+risk.">Whole embryo culture, teratogenesis, and the estimation of teratologic risk.</a> Teratology. 42, 635-642 (1990).</li><li>Matsuo, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mutation+in+the+Pax-6+gene+in+rat+small+eye+is+associated+with+impaired+migration+of+midbrain+crest+cells.">A mutation in the Pax-6 gene in rat small eye is associated with impaired migration of midbrain crest cells.</a> Nat Genet. 3, 299-304 (1993).</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>Yamamoto, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nodal+antagonists+regulate+formation+of+the+anteroposterior+axis+of+the+mouse+embryo.">Nodal antagonists regulate formation of the anteroposterior axis of the mouse embryo.</a> Nature. 428, 387-392 (2004).</li><li>Inoue, T., et al. <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+cadherins+in+maintaining+the+compartment+boundary+between+the+cortex+and+striatum+during+development.">Role of cadherins in maintaining the compartment boundary between the cortex and striatum during development.</a> Development. 128, 561-569 (2001).</li><li>Takahashi, M., 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+regulates+specification+of+ventral+neurone+subtypes+in+the+hindbrain+by+establishing+progenitor+domains.">Pax6 regulates specification of ventral neurone subtypes in the hindbrain by establishing progenitor domains.</a> Development. 129, 1327-1338 (2002).</li><li>Calegari, F., Haubensak, W., Yang, D., Huttner, W. B., Buchholz, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specific+RNA+interference+in+postimplantation+mouse+embryos+with+endoribonuclease-prepared+short+interfering+RNA.">Tissue-specific RNA interference in postimplantation mouse embryos with endoribonuclease-prepared short interfering RNA.</a> Proc Natl Acad Sci U S A. 99, 14236-14240 (2002).</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><li>Pryor, S. E., Massa, V., Savery, D., Greene, N. D., Copp, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convergent+extension+analysis+in+mouse+whole+embryo+culture.">Convergent extension analysis in mouse whole embryo culture.</a> Methods Mol Biol. 839, 133-146 (2012).</li><li>Kikkawa, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dmrta1+regulates+proneural+gene+expression+downstream+of+Pax6+in+the+mammalian+telencephalon.">Dmrta1 regulates proneural gene expression downstream of Pax6 in the mammalian telencephalon.</a> Genes Cells. 18, 636-649 (2013).</li><li>Tabata, H., Nakajima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+in+utero+gene+transfer+system+to+the+developing+mouse+brain+using+electroporation:+visualization+of+neuronal+migration+in+the+developing+cortex.">Efficient in utero gene transfer system to the developing mouse brain using electroporation: visualization of neuronal migration in the developing cortex.</a> Neuroscience. 103, 865-872 (2001).</li><li>Fukuchi-Shimogori, T., Grove, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neocortex+patterning+by+the+secreted+signaling+molecule+FGF8.">Neocortex patterning by the secreted signaling molecule FGF8.</a> Science. 294, 1071-1074 (2001).</li><li>Saito, T., Nakatsuji, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+into+the+embryonic+mouse+brain+using+in+vivo+electroporation.">Efficient gene transfer into the embryonic mouse brain using in vivo electroporation.</a> Dev Biol. 240, 237-246 (2001).</li><li>Turnbull, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound+backscatter+microscopy+of+mouse+embryos.">Ultrasound backscatter microscopy of mouse embryos.</a> Methods Mol Biol. 135, 235-243 (2000).</li><li>Pierfelice, T. J., Gaiano, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound-guided+microinjection+into+the+mouse+forebrain+in+utero+at+E9.5.">Ultrasound-guided microinjection into the mouse forebrain in utero at E9.5.</a> J Vis Exp. (45), e2047 (2010).</li><li>Quinlan, G. A., Khoo, P. L., Wong, N., Trainor, P. A., Tam, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+grafting+and+labeling+in+postimplantation+mouse+embryos.">Cell grafting and labeling in postimplantation mouse embryos.</a> Methods Mol Biol. 461, 47-70 (2008).</li><li>Nagy, A., Gertsenstein, M., Vinterstein, K., Behringer, R., Nagy, A., Gertsenstein, M., Vinterstein, K., Behringer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roller+culture+of+postimplantation+embryos.">Roller culture of postimplantation embryos.</a> Manipulating the Mouse Embryo: A Laboratory Manual. , 237-241 (2003).</li><li>Garcia, M. D., Udan, R. S., Hadjantonakis, A. K., Dickinson, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+rat+serum+for+culturing+mouse+embryos.">Preparation of rat serum for culturing mouse embryos.</a> Cold Spring Harb Protoc. 2011, 391-393 (2011).</li><li>Brown-Woodman, P. D., Ritchie, H. E., Korabelnikoff, A., Emmanuel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replacement+of+ether+with+alternate+volatile+anesthetics+for+collection+of+rat+serum+used+in+embryo+culture.">Replacement of ether with alternate volatile anesthetics for collection of rat serum used in embryo culture.</a> Toxicol In Vitro. 18, 719-724 (2004).</li><li>Gray, J., Ross, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+tube+closure+in+mouse+whole+embryo+culture.">Neural tube closure in mouse whole embryo culture.</a> J Vis Exp. (56), e3132 (2011).</li><li>Machado, C. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+phrenic+neuron+identity+in+embryonic+stem+cell-derived+motor+neurons.">Reconstruction of phrenic neuron identity in embryonic stem cell-derived motor neurons.</a> Development. 141, 784-794 (2014).</li><li>Glanville-Jones, H. C., Woo, N., Arkell, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+whole+embryo+culture+with+commercially+available+reagents.">Successful whole embryo culture with commercially available reagents.</a> Int J Dev Biol. 57, 61-67 (2013).</li><li>Ornoy, A., Yacobi, S., Yaffee, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+of+culture+of+11.5-day-old+rat+embryos+in+DMEM/F12+and+20%+fetal+bovine+serum.">A simple method of culture of 11.5-day-old rat embryos in DMEM/F12 and 20% fetal bovine serum.</a> J Anat. 203, 419-423 (2003).</li><li>Moore-Scott, B. A., Gordon, J., Blackburn, C. C., Condie, B. G., Manley, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+serum-free+in+vitro+culture+technique+for+midgestation+mouse+embryos.">New serum-free in vitro culture technique for midgestation mouse embryos.</a> Genesis. 35, 164-168 (2003).</li><li>Kalaskar, V. K., Lauderdale, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Embryonic+Development+in+a+Serum-free+Whole+Embryo+Culture+System.">Mouse Embryonic Development in a Serum-free Whole Embryo Culture System.</a> J Vis Exp. (85), e50308 (2014).</li></ol>

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><tbody><tr><td>Isoflurane</td><td>Abbott</td><td>B506</td><td>For rat anesthesia.</td></tr><tr><td>Large scissors</td><td>Napox</td><td>B-7H</td><td>Stelized scissors for cutting the skin and muscle of rats.&amp;nbsp;</td></tr><tr><td>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><td>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><td>Syringe (20 ml)</td><td>TERUMO</td><td>SS-29ESZ</td><td></td></tr><tr><td>Needle (21 G x 5/8&quot;)</td><td>TERUMO</td><td>NN-2116R</td><td></td></tr><tr><td>Sterile test (spitz) tube (10 ml)</td><td>ASIAKIZAI</td><td>1101C000B-10</td><td>For collection of boold</td></tr><tr><td>Sterile disposable pipette</td><td>Eiken Chemical</td><td>CD2000 No.4</td><td></td></tr><tr><td>Sterilie disposable tube (15 ml)&amp;nbsp;</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 ...

preparation-of-rat-serum-suitable-for-mammalian-whole-embryo-culture

Research

JoVE Journal

Biology

13.8K 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.

Video: Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

Developmental Biology

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 ...

Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, cell culture has been widely used in developmental biology studies. However, it is important to determine whether in vitro cultured cells truly represent in vivo cell types. Grafting cells into embryos, followed by an assessment of their contribution during development is a useful method to determine the potential of in vitro cultured cells. In this study, we describe a method for grafting cells into a defined site of early postimplantation mouse embryos, followed by ex vivo culture. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing precise localization and adjustment of the number of cells receiving exogenous DNA with both high transfection efficiency and low cell death. These techniques, which do not require any specialized equipment, render experimental manipulations of the gastrulation and early organogenesis-stage mouse embryo possible, allowing analysis of commitment in cultured cell subpopulations and the effect of genetic manipulations in situ on cell differentiation.

We demonstrate a method for grafting cultured cells into defined sites of early mouse embryos to determine their in vivo potential. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing the precise delivery of exogenous DNA into a few cells in the embryos.

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, ...

Preparation of Rat Oligodendrocyte Progenitor Cultures and Quantification of Oligodendrogenesis Using Dual-infrared Fluorescence Scanning

Efficient oligodendrogenesis is the therapeutic goal of a number of areas of research including spinal cord injury, neonatal hypoxia, and demyelinating diseases such as multiple sclerosis and transverse myelitis. Myelination is required to not only facilitate rapid impulse propagation within the central nervous system, but also to provide trophic support to underlying axons. Oligodendrocyte progenitor cells (OPCs) can be studied in vitro to help identify factors that may promote or inhibit oligodendrocyte differentiation. To date, many of the methods available to evaluate this process have either required large numbers of cells, thus limiting the number of conditions that can be investigated at any one time, or labor-intensive methods of quantification. Herein, we describe a protocol for the isolation of large numbers of highly pure OPCs together with a fast and reliable method to determine oligodendrogenesis from multiple conditions simultaneously. OPCs are isolated from P5-P7 neonatal rat cortices and grown in vitro for three days prior to differentiation. Four days after differentiation, oligodendrogenesis is evaluated using a dual-infrared fluorescence-scanning assay to determine expression of the myelin protein.

Oligodendrocytes are the myelinating cells of the central nervous system. This protocol describes a method for the isolation and culture of their precursors, oligodendrocyte progenitor cells, from rat cortices, as well as a fast and reliable quantitative method to evaluate oligodendrogenesis in vitro in response to experimental factors.

Efficient oligodendrogenesis is the therapeutic goal of a number of areas of research including spinal cord injury, neonatal hypoxia, and demyelinating ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Neuroscience

Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) would provide a powerful platform for neuroscientists to investigate axo-glial interactions, properties and differentiation of multipotent progenitors, and progression of oligodendroglial lineage cells at the cellular and molecular level.  We describe here a CNS aggregate culture system from embryonic rat forebrains, which can be maintained in a serum-free medium up to 3-4 weeks and is used in our laboratory as a model to study neuron-glia interaction and CNS myelination.  This video clip will demonstrate how to isolate and grow these CNS aggregate cultures from E16 rat brain.  Furthermore, from the same brain dissection, highly enriched regular dissociated neuronal cultures can be readily obtained and used for various studies on CNS neurons or used for co-cultures with other cells. 

A protocols for an embryonic rat brain aggregate culture system is described. Multipotent progenitors in the aggregates can develop and differentiate into neurons, astrocytes and oligodendrocytes.

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) ...

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. ...

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

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

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

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

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 ...

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Summary

Explore More Videos

Chapters in this video

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

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

Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

Preparation of Rat Oligodendrocyte Progenitor Cultures and Quantification of Oligodendrogenesis Using Dual-infrared Fluorescence Scanning

Isolation of Murine Embryonic Hemogenic Endothelial Cells

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

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

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