The work was supported by grants from the Universit&#233; catholique de Louvain (Action de recherch&#233; concert&#233;es) and the Fund for Scientific Medical Research (F.R.S.-FNRS, Belgium). A.-S.D. is a doctoral fellow from T&#233;l&#233;vie, A.-C.H. held a fellowship from the Fonds pour la formation &#224; la Recherche dans l'Industrie et dans l'Agriculture (Belgium), M.V. is supported by the de Duve Institute and C.E.P. is a senior research associate of the F.R.S.-FNRS (Belgium).

Mice were raised and treated according to the principles of laboratory animal care of the University Animal Welfare Committee. All procedures and protocols were approved by this Committee.
1. Coating of Microscopy Plastic Culture Chambers
NOTE: Perform all the following steps under sterile conditions in a laminar flow hood. The two types of coating are functionally equivalent.
<ol>
	<li>Coat plastic culture chambers with fibronectin one day prior to isolation of thyroid tissues:
	<ol>
		<li>Dilute sterile fibronectin to a 50 &#956;g/ml final concentration in M199 medium supplemented with 100 U/ml penicillin, 100 &#956;g/ml streptomycin, 0.25 &#956;g/ml fungizone and 2 mM glutamine12.</li>
		<li>Cover the wells with 200 &#956;l of diluted fibronectin and incubate overnight in a 4 &#176;C refrigerator.</li>
		<li>On the day of the dissection, aspirate the fibronectin, rinse the wells once with M199 and add a minimum volume of 200 &#956;l of M199 supplemented with antibiotics, glutamine and 10% fetal calf serum.</li>
		<li>Leave the coated chambers in the incubator until ready to plate the explants.</li>
	</ol>
	</li>
	<li>Coat plastic culture chambers with type I collagen 2 hr before isolation of thyroid tissues:
	<ol>
		<li>Dilute sterile type I collagen to a 50 &#956;g/ml final concentration in 10 mM HCl. NOTE: Keep collagen stock solution on ice.</li>
		<li>Cover the wells with 200 &#956;l of diluted type I collagen and incubate for 2 hr at room temperature (RT).</li>
		<li>Aspirate the type I collagen solution, rinse the wells once with M199 and add a minimum volume of 200 &#956;l of M199 supplemented with antibiotics, glutamine and 10% fetal calf serum.</li>
		<li>Leave the coated chambers in the incubator until ready to plate the explants.</li>
	</ol>
	</li>
</ol>
2. Dissection of Thyroid Anlagen-containing Tissues (e12.5) or Thyroid Lobes (e13.5-e14.5)
NOTE: Use clean dissecting tools and Petri/culture plates. The general steps and incisions described below are the same for e12.5 and e14.5 embryos.
<ol>
	<li>Sacrifice timed-pregnant female mice at the desired embryonic (e) day (from e12.5 to e14.5) and remove the uterus using dissecting instruments.</li>
	<li>Place the dissected uterus in a 10&#160;cm plate containing HBSS.</li>
	<li>Hold one extremity of the uterus with forceps and open the uterus with small scissors to liberate progressively all the embryos.</li>
	<li>Separate the embryos from the placenta with scissors and transfer the embryos with their extraembryonic membranes in a new 10&#160;cm plate containing HBSS.</li>
	<li>Remove the yolk sac and the amnion. NOTE: From this point, work under a stereomicroscope with transillumination from below; increasing magnification with dissection steps. Use tungsten needles in glass capillaries as dissecting tools.</li>
	<li>Using the needles like a knife and fork, remove the upper part of the head, including the upper jaw and the ears (Figure&#160;1A, cut along a).</li>
	<li>Hold the embryo on its back and remove first an extra piece of the neural tube (Figure&#160;1A, cut along b), then the lower part of the embryo, just under the anterior limbs and above the heart (Figure&#160;1B).</li>
	<li>Transfer the embryo slice containing the tongue (to orient the tissue) and neck region in a new 10&#160;cm plate containing HBSS.</li>
	<li>Remove the neural tube and tissues posterior to the esophagus/trachea (Figure 1C, cut along a).</li>
	<li>Gently section the tissue slice along both sides of the tongue (Figure&#160;1C, cut along b and b&#8217;).</li>
	<li>Locate the esophagus and trachea, in the prolongation of the tongue, and dissect away the tongue, leaving the arytenoid swelling (Figure&#160;1D, cut along a), and the tissue on both sides of the esophagus/trachea (Figure&#160;1D, cut along b and b&#8217;).</li>
	<li>Remove the esophagus, and place the trachea with its ventral side facing up.</li>
	<li>Dissect away the undesired tissues such as the thymus (Figure&#160;1E) and all the tissues located laterally to the pharyngeal arch arteries. For e13.5 or e14.5 embryos, visualize the thyroid lobes on each side of the trachea (Figure&#160;1F; red dotted lines) and further dissect the lobes from the trachea.</li>
	<li>Transfer the isolated e12.5 trachea region, or the e13.5-e14.5 thyroid lobes into a 35&#160;mm culture plate containing pre-warmed M199 medium supplemented with antibiotics, glutamine and 10% fetal calf serum. Transfer explants using a prewetted filter tip on a P-200 micropipette. For e12.5 explants, cut the extremity of the tip to widen the opening. NOTE: Routinely, and if embryos have the same genotype, perform steps 6 &#38; 7 on all the embryos, then 9-11, and finally 12 &#38; 13.</li>
</ol>
3. Plating and Culture of Thyroid Explants
<ol>
	<li>Wash the explants by successive transfer in three wells of a 24-well plate containing 1 ml of pre-warmed culture medium (= M199 supplemented with antibiotics, glutamine and 10% fetal calf serum).</li>
	<li>Plating of thyroid lobes on coated microscopy plastic culture chambers:
	<ol>
		<li>Aspirate M199 culture medium from the coated chambers.</li>
		<li>Carefully, transfer the thyroid explants into the culture chambers using a micropipette and filter tips. Place one explant in the center of each chamber.</li>
		<li>Remove excess medium and place the culture chamber in a tissue culture incubator (37 &#176;C; 5% CO2) for 2 hr to let the explants attach to the matrix.</li>
		<li>Gently add 200 to 300 &#956;l of pre-warmed culture medium to each well.</li>
		<li>Incubate for long-term culture in the tissue culture incubator.</li>
	</ol>
	</li>
	<li>Plating of trachea region or thyroid lobes on semiporous filters:
	<ol>
		<li>Fill the number of wells of a 24-well plate required with 330 &#956;l of pre-warmed culture medium.</li>
		<li>Place filters (e.g. 0.4 &#181;m Millipore filter) on the medium, taking care to avoid trapping of air bubbles below the culture filter.</li>
		<li>Carefully, using a micropipette and filter tips, transfer the thyroid explants with a minimal volume of medium onto the center of the filters (maximum 4/filter).</li>
		<li>Remove the excess of medium and space out the explants with a tungsten needle.</li>
		<li>Incubate for long-term culture in the tissue culture incubator. NOTE: Change culture medium every other day under the laminar flow hood. Culture the explants up to 7 days.</li>
	</ol>
	</li>
</ol>
4. Whole-mount Immunofluorescence Staining Protocol for Thyroid Explants
Dislodge explants grown on filters (after step 2 below) using tungsten needles or fine forceps and transfer in a 24-well plate for all the steps, except for primary (step 5) and secondary (step 7) antibody incubations (96-well plate). For adherent explants cultured on microscopy plastic chambers, perform all the steps in the culture chambers.
<ol>
	<li>Aspirate M199 culture medium and fix the explant with ice-cold 4% paraformaldehyde (PFA) for 20 min (500 &#181;l in 24-well dish and 300 &#181;l in microscopy slides).</li>
	<li>Wash 2x 10 min in Tris Buffered Saline with Triton (TBST; 50 mM Tris HCl pH 7.5, 150 mM NaCl, 0,1% Triton X-100) at RT.</li>
	<li>Process the explants immediately or store at -20 &#176;C. NOTE: For long-term storage at -20 &#176;C, dehydrate the explants in methanol by gradually increasing methanol concentration in TBST to reach 100% methanol. When ready to continue with the immunostaining (step 4), rehydrate the explants by gradually adding TBST to methanol.</li>
	<li>Replace TBST with the blocking solution (10% normal Goat Serum in TBST) and incubate on a sea-saw rocker for 30-60 min at RT.</li>
	<li>Dilute primary antibody in blocking solution (100-200 &#181;l per condition). Transfer the &#8220;filter&#8221; explants from a 24-well plate to a 96-well plate. Incubate explants with the primary antibodies on a sea-saw rocker overnight at 4 &#176;C.</li>
	<li>The next day, discard the primary antibody solution, transfer the &#8220;filter&#8221; explants back in a 24-well plate, and wash at least five times with TBST on a sea-saw rocker for 45-60 min each at RT.</li>
	<li>Dilute secondary antibody (e.g. Goat anti-X-Alexa Fluor at 1:2,000 dilution) in TBST containing 1% of normal goat serum and incubate the explants with this dilution overnight on a sea-saw rocker at 4 &#176;C and in the dark. For nuclear counterstaining, include 1 &#956;g/ml of bis-Benzimide (Hoechst) together with the secondary antibody solution.</li>
	<li>The next day, discard the secondary antibody solution and wash 5x with TBST on a sea-saw rocker for 45-60 min each at RT. During the washes, keep the explants in the dark.</li>
	<li>Perform an additional wash in TBST gently swirling overnight at 4 &#176;C in the dark.</li>
	<li>Postfix the immunostained explants in 4% PFA at 4 &#176;C for 10 min.</li>
	<li>Wash 2x&#160;10 min with TBST.</li>
	<li>Transfer the explants either gradually in methanol 100% for long-term storage at -20 &#176;C, or analyze immediately. For explants cultured on filters, and immunostained in a 24-well plate, transfer in a microscopy chamber (e.g. LabTek or Ibidi) with TBST or better fluorescent mounting medium prior to analysis with a Multiphoton or confocal microscope.</li>
</ol>
5. Extraction of RNA from Thyroid
Work in an RNase-free environment. Therefore, use nucleic acid and nuclease free pipette tips and clean both bench and equipment from contaminants and RNases. This protocol being based on phenol/chloroform, perform the first steps (1 to 7) under a chemical hood.
<ol>
	<li>Homogenize one thyroid explant or two thyroid lobes with 300 &#181;l of RNA extraction solution using a disposable pestle&#160;for 1.5 ml tube. In between samples, wash the pestle in 2% SDS, then water, then ethanol.</li>
	<li>Add another 100 &#181;l of RNA extraction solution, vortex 10 sec and incubate at RT for 10 min.</li>
	<li>Vortex for 30 sec and add 20 ng of tRNA to each sample.</li>
	<li>Vortex for 1 min and add 80 &#181;l of chloroform.</li>
	<li>Vortex vigorously for 30 sec and incubate 15 min at RT.</li>
	<li>Spin for 15 min at 19,000 x g in a refrigerated (4 &#176;C) centrifuge.</li>
	<li>Transfer carefully the colorless upper aqueous phase in a fresh tube containing 20 &#956;g of glycogen as coprecipitant.</li>
	<li>Vortex and add 200 &#181;l of 100% isopropanol alcohol.</li>
	<li>Vortex vigorously for 15-30 sec and let RNA precipitate at -20 &#176;C for 2-3 hr or at -80 &#176;C for 1 hr.</li>
	<li>Spin for 30 min at 19,000 x g at 4 &#176;C, and eliminate supernatant.</li>
	<li>Wash the pellet with 450 &#181;l of cold 75% ethanol and vortex to dislodge the pellet.</li>
	<li>Spin for 10 min at 19,000 x g at 4 &#176;C, and eliminate supernatant.</li>
	<li>Dry the pellet under the hood for 5 min.</li>
	<li>Resuspend the pellet with 10 &#181;l of RNase-free water and incubate 10 min at RT.</li>
	<li>Assay RNA concentration, store at -80 &#176;C or reverse transcribe for gene expression analysis.</li>
</ol>

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The authors have nothing to disclose.

This paper describes a method for dissecting and culturing thyroid explants (e12.5)&#160;or lobes (e14.5) in order to study and better understand the complex events leading to thyroid formation. Due to the dual origin of the thyroid anlagen (the midline anlage and the two lateral ultimobranchial bodies), and their small size, a fragment of e12.5 tissue localized rostral to the pharyngeal arch arteries, and containing the trachea, but not the esophagus is microdissected. As illustrated, within a period of four days, this ...

The thyroid gland is a collection of independent epithelial spheres, called follicles, surrounded by a dense network of endothelial capillaries. This organization allows thyroid function: endothelial capillaries provide thyrocytes with iodine, required for T3 and T4 hormone synthesis, and distribute these latter to the whole body. Scattered in between the follicles and capillaries, C-cells produce the hypocalcemic hormone calcitonin1. Although adult thyroid architecture and functions are well known, the cellular and molecular mechanisms involved in thyroid embryonic development (follicle formation and differentiation) are far from being understood.
During embryogenesis, thyrocytes progenitor originates as a thickening (the midline anlage) of the ventral wall of the foregut endoderm at embryonic day (e) 8.5 in the mouse embryo, while C-cells progenitors originate at e11.5 as droplet-shaped protrusions (the ultimobranchial bodies) of the fourth pharyngeal pouches2-6. The midline bud then detaches from the endoderm, expands bilaterally to fuse at e13.5 with the ultimobranchial bodies on each side of the trachea. Finally, thyrocytes organize into follicles and C-cells differentiate.
Current knowledge on thyroid formation mainly comes from histological analysis of fixed tissues, but the morphogenetic events involved in thyroid formation are highly dynamic and involve communications and interactions between different cell types and with the extracellular matrix. Recent work showed that thyrocyte progenitors produce high levels of VEGF to recruit endothelial cells to the developing thyroid, and, in turn, recruited endothelial cells promote follicle formation and C-cells differentiation7.
Most ex vivo studies on the thyroid have been performed on isolated adult thyroid-derived cells, grown either on 2D tissue culture plastic dishes or in 3D matrices. Using these types of cultures, differentiated follicular cells either remain polarized and organized as follicles or reacquire a 3D organization8-10. However, these pure epithelial cells, explanted from their physiological environment, and cultured in 2D, ignore interactions with extracellular matrix, cytokines, growth factors and with other cell types such as the endothelial or nerves cells that they normally encounter in vivo. A very nice study recently described a differentiation protocol of ES cells into thyroid follicles using a final culture step in 3D matrigel11. However, these 3D cultures lack contact with other cell types.
Based on previous expertise on pancreas and salivary glands organ culture12-14, a method for dissecting mouse embryonic thyroid anlagen and culturing the explants on semiporous filters or on microscopy plastic slides was developed.
When working with e12.5 embryos, the dual origin of the thyroid anlagen (the midline anlage and the two lateral ultimobranchial bodies) imposed the microdissection of a large fragment of tissue. This contained the trachea, but not the esophagus, and extended from the pharyngeal arch arteries up to the arytenoid swelling. When cultured on filters, the midline anlage extends laterally on each side of the trachea, where they fuse with the ultimobranchial bodies to form the two thyroid lobes, still connected by a narrow isthmus.
In culture, epithelial cells proliferate, organize into follicles and differentiate into thyrocytes and C-cells, depending on their origin. Endothelial cells contained in the microdissected tissue also proliferate and invade the thyroid lobes to finally associate closely with the epithelial follicular structure, independently of blood flow or circulating factors. As development of the explants faithfully recapitulates in vivo development, this culture system is optimal to study morphogenetic and differentiation events occurring during thyroid development.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos, and the culture system is amenable to loss- and gain-of-function experiments. Finally, time-lapse imaging of fluorescently labeled thyroid explants on microscopy plastic dishes could be exploited to better investigate the kinetics and continuity of morphogenetic movements that occur in vivo. Time-lapse imaging has already been used to study branching morphogenesis of the pancreas15,16 or the ureteric bud17.

<table border="0" cellpadding="0" cellspacing="0" width="698">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">&#181;-slide 8 well, ibiTreat, tissue culture treated, sterile</td>
			<td style="width:85px;">proxylab</td>
			<td style="width:119px;">80826</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagen Type I, rat tail</td>
			<td style="width:85px;">Millipore</td>
			<td>08-115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fibronectin from human plasma</td>
			<td style="width:85px;">Invitrogen</td>
			<td># 33010-018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">M199</td>
			<td style="width:85px;">Invitrogen</td>
			<td>31150-022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">HBSS</td>
			<td style="width:85px;">Invitrogen</td>
			<td>14025-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Tungsten wire</td>
			<td style="width:85px;">Goodfellow</td>
			<td style="width:119px;">LS237450</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">culture plate insert (12 mm diameter)</td>
			<td style="width:85px;">Millipore</td>
			<td>PICM01250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">GlycoBlue</td>
			<td style="width:85px;">Invitrogen</td>
			<td style="width:119px;">AM9516</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">E-cadherin</td>
			<td style="width:85px;">BD Biosciences</td>
			<td style="width:119px;">610182</td>
			<td>1/1000</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Ezrin</td>
			<td style="width:85px;">Thermo Scientific</td>
			<td style="width:119px;">MS-661-P1</td>
			<td>1/400</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">PECAM</td>
			<td style="width:85px;">BD Biosciences</td>
			<td style="width:119px;">550274</td>
			<td>1/100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Hoechst</td>
			<td style="width:85px;">Sigma</td>
			<td style="width:119px;">B2261</td>
			<td></td>
		</tr>
	</tbody>
</table>

an ex vivo culture system to study thyroid development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

Thyroid anlages (midline and UB, together with surrounding tissues), and thyroid lobes are dissected from mouse embryos at e12.5 and e13.5/e14.5, respectively (Figure 1). After one day in culture on filter, the midline anlage is visible (Figure 2A) as an elongated tissue spanning on top of the trachea. It progressively forms two lobes on each side of the trachea. If isolated thyroid lobes (e13.5 or e14.5) are cultured, they will expand, undergo morphogenesis and epithelial cells will org...

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An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

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11.4K Views.  Université catholique de Louvain & de Duve Institute. This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

Video: An Ex vivo Culture System to Study Thyroid Development

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

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

Method for Culture of Early Chick Embryos ex vivo New Culture

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

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

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 ...

Closed System Cell Culture Protocol Using HYPERStack Vessels with Gas Permeable Material Technology

Large volume adherent cell culture is currently standardized on stacked plate cell growth products when microcarrier beads are not an optimal choice. HYPERStack vessels allow closed system scale up from the current stacked plate products and delivers >2.5X more cells in the same volumetric footprint. The HYPERStack vessels function via gas permeable material which allows gas exchange to occur, therefore eliminating the need for internal headspace within a vessel. The elimination of headspace allows the compartment where cell growth occurs to be minimized to reduce space, allowing more layers of cell growth surface area within the same volumetric footprint.
For many applications such as cell therapy or vaccine production, a closed system is required for cell growth and harvesting. The HYPERStack vessel allows cell and reagent addition and removal via tubing from media bags or other methods.
This protocol will explain the technology behind the gas permeable material used in the HYPERStack vessels, gas diffusion results to meet the metabolic needs of cells, closed system cell growth protocols, and various harvesting methods.

An Introduction into the technology, protocol and handling of the Corning HYPERStack Vessels and accessories used for high yield adherent cell culture. The protocol will show how to use the closed system vessels for increasing cell harvesting over current stacked plate products.

Large volume adherent cell culture is currently standardized on stacked plate cell growth products when microcarrier beads are not an optimal choice.   ...

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

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

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

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

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

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...

Establishment of an Extracellular Acidic pH Culture System

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

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

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