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>
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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. 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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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13.8K Aufrufe.  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.

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

Organotypic Slice Culture of E18 Rat Brains

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, organotypic slice cultures allow one to perform a number of manipulations that are not presently feasible in-vivo. To date, organtotypic embryonic brain slice cultures have been used to follow individual cells using time-lapse microscopy, manipulate the expression of genes in the ganglionic emanances (a region that is hard to target by in-utero electroporation), as well as for pharmacological studies. In this video protocol we demonstrate how to make organotypic slice cultures from rat embryonic day 18 embryos. The protocol involves dissecting the embryos, embedding them on ice in low melt agarose, slicing the embedded brains on the vibratome, and finally plating the slices onto filters in culture dishes. This protocol is also applicable in its present form to making organotypic slice cultures from different embryonic ages for both rats and mice. 

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, ...

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

Preparation and Culture of Rat Lens Epithelial Explants for Studying Terminal Differentiation

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  Following division, these cells migrate posteriorly, where FGF diffusing from the retina induces them to differentiate into a posterior array of elongated lens fiber cells, which compose the bulk of the lens.  Differentiation of lens epithelial cells into lens fibers can be induced in vitro by culturing explants of the central region of the anterior epithelium in the presence of FGF-2.  Explants are prepared from lenses of neonatal rats by removing the lens from the eye and grasping the lens capsule on the posterior side with dissecting tweezers.  The posterior capsule is then gently torn open and pressed down into the plastic bottom of a tissue culture dish. The peripheral regions of the explant are removed with a scalpel and the central area is then cultured in the presence of 100ng/ml FGF-2 for as long as 2-3 weeks, depending on the parameters to be studied.  Since epithelial cells in cultured explants differentiate in approximate synchrony over a period of days to weeks, the time course of signaling and gene expression can be determined using molecular, biochemical, and pharmacological techniques.  Immunofluorescence microscopy is a powerful adjunct to these methods as it demonstrates the subcellular localization of proteins of interest and can reveal the physiological consequences of experimental manipulations of signaling pathways.

Explants of the central region of rat lens epithelia differentiate synchronously when cultured in the presence of FGF-2. Immunofluorescence microscopy of such cultures can provides novel information about gene expression and signaling events associated with terminal differentiation.

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  ...

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

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

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

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

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

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

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

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

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

Scale-Up of Mammalian Cell Culture using a New Multilayered Flask

A growing number of cell-based applications require large numbers of cells. Usage of single layer T-flasks, that are adequate during small-scale expansion, may become cumbersome, laborious and time-consuming when large numbers of cells are required. To address this need, the performance of a new multi-layered cell culture vessel to facilitate easy scale up of cells from single layered T-flasks will be discussed. The flasks tested are available in 3- and 5-layer format and enable culture and complete recovery of three and five times the number of cells respectively, compared to T-175 flasks. A key feature of the BD Multi-Flask is a mix/equilibration port that allows rapid in-vessel mixing as well as uniform distribution of cells and reagents within and 
between layers of each vessel and consistently produce cells that can be cultured in an environment that is congruent to T-175 flasks.
The design of these Multi-Flasks also allows for convenient pipette access for adding reagents and cells directly into the flasks as well as efficient recovery of valuable cells and reagents and reduces risk of contamination due to pouring. For applications where pouring is preferred over pipetting, the design allows for minimal residual liquid retention so as to reduce wastage of valuable cells and reagents.

Cells play an instrumental and increasing role in research, and the discovery and development of new therapeutics. With this increasing need for greater number of cells we need more efficient and effective ways for growing and harvesting attachment dependent cells. A Multilayered flask with the right features can serve this purpose.

A growing number of cell-based applications require large numbers of cells. Usage of single layer T-flasks, that are adequate during small-scale ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

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 ...

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is an excellent model for developmental biology research. During embryogenesis, zebrafish develop with a yolk mass, which presents three-dimensional challenges for sample observation and analysis. This protocol describes how to create two-dimensional flat mount preparations of whole mount in situ (WISH) stained zebrafish embryo specimens.

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...

Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without breaching the collagenous basement membrane. While DCIS is a non-obligate precursor to invasive breast cancers, the molecular mechanisms and cell populations that permit progression to invasive cancer are not fully known. To determine if progenitor cells capable of invasion existed within the DCIS cell population, we developed a methodology for collecting and culturing sterile human breast tissue at the time of surgery, without enzymatic disruption of tissue.
Sterile breast tissue containing ductal segments is harvested from surgically excised breast tissue following routine pathological examination. Tissue containing DCIS is placed in nutrient rich, antibiotic-containing, serum free medium, and transported to the tissue culture laboratory. The breast tissue is further dissected to isolate the calcified areas. Multiple breast tissue pieces (organoids) are placed in a minimal volume of serum free medium in a flask with a removable lid and cultured in a humidified CO2 incubator. Epithelial and fibroblast cell populations emerge from the organoid after 10 - 14 days. Mammospheres spontaneously form on and around the epithelial cell monolayer. Specific cell populations can be harvested directly from the flask without disrupting neighboring cells. Our non-enzymatic tissue culture system reliably reveals cytogenetically abnormal, invasive progenitor cells from fresh human DCIS lesions.

Primary cell culture using intact tissue organoids provides a model system that mimics the multi-cellular in vivo microenvironment. We developed a serum-free primary breast epithelium tissue culture model that perpetuates mixed cell culture lineages and exhibits differentiated morphology, without enzymatic tissue disruption. Breast organoids remain viable for &#62;6 months.

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without ...