Research in the Spagnoli lab. is funded by the Helmholtz Association, FP7-IRG-2008-ENDOPANC grant and ERC-2009-Starting HEPATOPANCREATIC Grant.

The protocol described here has been adapted from the technique originally described in Percival and Slack9 and optimized for confocal microscopy.
1. Coating of Glass Bottom Culture Dishes
The following steps should be carried out under sterile conditions in a laminar flow hood.
<ol>
 <li>Pancreatic explants are cultured in 35-mm Petri dishes with 20-mm diameter glass microwell bottom (e.g. MatTek Corporation). Use one glass bottom dish per explant culture and for the whole duration of the culture. On the day before the dissection and isolation of the pancreatic buds, coat the glass bottom microwells with fibronectin.</li>
 <li>Dilute sterile fibronectin in cell culture-grade water to a 50 &mu;g/ml final concentration and add a minimum volume of it (~150 &mu;l) into the 20-mm microwell of the MatTek plates covering the whole glass surface (Figure 1E). Place the dishes in a 4 &deg;C refrigerator for overnight incubation.</li>
 <li>On the day of the dissection, aspirate the fibronectin, rinse the microwell with cell culture-grade water and add a minimum volume of 150 &mu;l of BME (Basal Medium Eagle) medium supplemented with Pen/Strep (1%), Glutamine (1%) and 50 &mu;g/ml Gentamicin [Dissection Medium]. Leave the coated dishes in the laminar flow hood until ready to plate the explants.</li>
</ol>
If not all fibronectin-coated dishes are used, they can be stored for up to 1 week at 4 &deg;C. Do not let the dishes dry, fill them with Dissection Medium and place them in the refrigerator.
In addition to fibronectin, pancreatic explants have been successfully cultured on various substrates, such as laminin9, matrigel10,11 or microporous membranes12. Because of its effects on branching, we use fibronectin9 as substrate.
2. Dissection of Dorsal Pancreatic Bud from E11.5 - E12.5 Mouse Embryos
<ol>
 <li>Timed-pregnant female mice at embryonic day (E) 11.5 or 12.5 are sacrificed and embryos are collected using dissecting instruments previously cleaned with 70% Ethanol. Place the dissected uterus in 10-cm plates containing ice-cold PBS supplemented with 50 &mu;g/ml gentamicin.</li>
 <li>Under a stereomicroscope with illumination from above, separate the uterus into individual embryo segments using small scissors or Dumont forceps, gently peel away the muscle of the uterus and remove the individual embryos, which are surrounded by the decidua. Use standard embryo dissection techniques13 to expose the embryos and remove the yolk sac and amnion. Subsequently, remove the upper body of the embryo (above the liver) and the tail region. This will allow exposure of inner organs (Figure 1B). Transfer the mid-body of the embryos into 10-cm plates containing ice-cold BME dissection medium.</li>
 <li>Now use only transillumination from below to visualize and to dissect the dorsal pancreatic bud under the stereomicroscope. Gently, open the mid-body of the embryo by making a lateral incision with forceps. Locate the stomach and the spleen. The dorsal pancreatic bud is attached laterally to the posterior region of the stomach (Figure 1C). Using Dumont forceps, dissect away the dorsal pancreatic bud from adjacent structures, such as the stomach and the spleen, but leaving some mesenchyme around the epithelium (Figure 1D). Using a glass Pasteur pipette, transfer the isolated bud into a 35-mm Petri dish containing cold BME dissection medium supplemented with 10% fetal calf serum [Culture Medium]. Repeat these steps until all pancreata are isolated. If pancreata of different genotypes are isolated, keep them separate using Nunc 4-well plates.</li>
</ol>
3. Plating and Culture of Pancreatic Explants
Final plating of the explants is carried out in tissue culture room and/or in close proximity to the tissue culture incubator to minimize physical movement of the cultures.
<ol>
 <li>Under sterile conditions in a laminar flow hood replace the BME Dissection Medium in the fibronectin-coated MatTek dishes with ~150 &mu;l of BME culture medium pre-warmed at 37 &deg;C.</li>
 <li>Carefully, transfer the pancreatic explants into coated MatTek dishes (one explant per dish) for long-term culture using a glass Pasteur pipette. To ensure spreading during culture, the mesenchyme surrounding the explants can be slightly ripped, if necessary, with a fine needle.</li>
 <li>Gently, place the cultures in a tissue culture incubator (37 &deg;C; 5% CO2) for few hours to let the explants attach to the glass bottom microwells. Once they are attached, fill up the MatTek dishes with 1.5 ml to 2 ml of BME culture medium pre-warmed at 37 &deg;C. The day of dissection is referred to as day 0.</li>
 <li>Manipulation of the pancreatic explant cultures is carried out under sterile conditions in a laminar flow hood. The BME culture medium is changed every other day, unless experimental requirements dictate different timing (e.g. incubation of explants with chemical agents). The explants can be cultured in this condition for 5 days up to one week.</li>
</ol>
4. Whole-mount Immunofluorescence Staining Protocol for Pancreatic Explants
All steps of the immunofluorescence protocol (from fixation to imaging) are carried out in the same MatTek dish, which yields better images than a plastic-bottomed dish. After fixation, pancreatic explant cultures do not need to be kept under sterile conditions.
<ol>
 <li>Aspirate BME culture medium and wash the explant once with 2 ml 1X PBS.</li>
 <li>For explant fixation replace PBS with 2 ml ice-cold 4% paraformaldehyde (PFA) and place the dish on ice for 20 min. Swirl the plate gently from time to time, but avoid rocking platforms, as the explant might detach.</li>
 <li>Remove PFA and wash the explant three times with 2 ml of 1X PBS for 10 min each at room temperature (RT).</li>
 <li>Block with 2 ml blocking solution (1X PBS + 0.1% Triton-X + 3% Donkey Serum) for at least 30 min at RT.</li>
 <li>Dilute primary antibody in blocking solution and add ~150 &mu;l of the dilution directly into the 20-mm microwell of the MatTek plates covering the whole explant surface for overnight incubation at 4 &deg;C. To minimize physical movements, add primary antibody to the explants directly in the cold room (4 &deg;C). To prevent evaporation, before adding the antibody, place the MatTek plates in a humid chamber.</li>
 <li>The next day remove the antibody solution and wash three times with 2 ml 1X PBS + 0.1 % Tween-20 (PBT) for 30 min each at RT.</li>
 <li>Dilute secondary antibody in blocking solution [according to manufacturer's recommendations; we use donkey Alexa Fluor dyes secondary antibodies at 1:750 dilution] and add ~150 &mu;l of the dilution directly into the 20-mm microwell of the MatTek plates, covering the whole explant surface, for 1-2 hr incubation at RT in the dark. For nuclear counterstaining, include Hoechst together with secondary antibody dilution. To prevent evaporation, before adding the antibody, place the MatTek plates in a humid chamber.</li>
 <li>Wash three times with 2 ml 1X PBS + 0.1 % Tween-20 (PBT) for 30 min each at RT.</li>
 <li>Before mounting, wash once with 1X PBS to remove Tween-20. Mount by adding one drop of aqueous mounting medium (with anti-fading) into the 20-mm microwell of the MatTek plates. Gently, cover the explant with a glass coverslip. Avoid air bubbles.</li>
 <li>Explants on MatTek plates can be viewed under standard phase contrast microscopy or confocal microscopy, depending on the goals of the experiment. For confocal microscopy analysis we use a Zeiss LSM 700 inverted setting. Set up appropriate lasers for fluorophores used. Select an objective that will image the desired area of the pancreatic explant. A 10X water-immersion objective is suitable to keep the entire explant in the field of view. A 40X water- or oil-immersion objectives are suitable to focus on specific regions of the pancreatic explant with greater resolution. The explants can be optically sectioned and then the acquired Z-stacks reconstructed in 3D, using appropriate software.</li>
</ol>
5. Live-cell Imaging Protocol of Pancreatic Explants
<ol>
 <li>Environmental chamber setup: use an environmental enclosure and a small incubation chamber [with temperature and CO2 control] that sits on the microscope stage. Proper culturing of the embryonic pancreas requires a stable temperature of 37 &deg;C and a controlled atmosphere of humidified 5% CO2. Suitable chambers are available from several vendors (e.g., Zeiss, Nikon, Olympus). Assemble the heater box and turn it on at least 1 hr before imaging starts to allow the equipment to warm up and equilibrate CO2 content to 5%.</li>
 <li>For time-lapse imaging of pancreatic explant cultures, we use a Zeiss LSM 700 inverted microscope. For lasers and objective selection see point 4.10.</li>
 <li>Let the pancreatic explant attach well to the glass-bottom dish before starting the live imaging. [We usually perform live imaging starting on Day 1]. 
 In a laminar flow hood, aspirate and replace the explant medium with fresh BME culture medium to start with optimal nutrient conditions.</li>
 <li>Gently transfer the explants to the microscope room and place the MatTek dish in the incubation chamber into the specimen holder of the confocal microscope.</li>
 <li>Prepare the objective (e.g. add oil or water-medium according to the choice of immersion objective) and focus on the region of interest in the explant. Close the environmental chamber properly. To prevent evaporation during time-lapse acquisition with water-immersion objectives, in place of water it is recommended to use viscous immersion fluids that are commercially available (e.g. Zeiss Immersol W).</li>
 <li>Set up laser intensity, Z-stack acquisition parameters and time interval (see also Discussion for Troubleshooting). Start the experiment.</li>
 <li>After imaging, export Z stacks and all time points and analyze them using appropriate software (e.g. ImageJ, Volocity, Imaris, Huygens).</li>
</ol>
6. Representative Results
Dorsal pancreatic buds (together with the surrounding mesenchyme) were dissected from mouse embryos between E11.5 and E12.5 and plated directly on glass bottom culture dishes (Figure 1). Blood vessels are present in the mesenchyme surrounding the epithelium and can be visualized by immunostaining for the endothelial marker Pecam12 (data not shown). After two days of culture, pancreatic explants underwent significant growth and started to organize themselves into branched epithelial structures, whose nuclei were positive for the pancreatic transcription factor, Pdx1 (Figure 2A-C). The average z-thickness of pancreatic explants on day 3 of culture is 60-80 &mu;m. The surface and volume of the explants can be measured using appropriate softwares (e.g. Huygens). Morphology and immunofluorescence analyses showed that pancreatic explants cultured using this protocol recapitulated in vivo early pancreatic differentiation and morphogenetic events5,10,11,14 (Figure 2). Pancreatic explants on day 4 of culture underwent typical "tip and trunk" segregation of the epithelium8, displaying cpa1+ progenitor cells at the tips of the epithelial branches (Figure 2C) and differentiated insulin+ and glucagon+ cells organized as clusters inside the trunk (Figure 2D). In addition, the pancreatic epithelium in the ex vivo cultures showed proper cytoskeleton organization and cell polarity, with apical localization of F-actin (Figure 2E, in green) and b1-integrin at the basal membranes (Figure 2E, in red).
To study pancreatic branching in realtime,embryonic pancreatic explants were isolated from the dual-color fluorescent reporter mouse strain membrane-Tomato/membrane-Green15 (mT/mG; mouse strain is available at the Jackson Laboratory) (Figure 3). Still frames from a time-lapse movie of mT/mG pancreatic explants grown in culture are shown in Figure 3. The localization of the mT fluorescent protein to membrane structures outlines cell morphology and allows resolution of fine cellular processes, enabling to track cell remodeling and migration over time.
After a 12-hr time-lapse experiment the mT fluorescence signal remains viable and detectable, even though its intensity is reduced, most likely due to photodamage (Figure 3). Technical solutions to avoid or reduce photodamage and phototoxicity are discussed in the Discussion section.
<img alt="Figure 1" src="/files/ftp_upload/3979/3979fig1.jpg" /> 
Figure 1. Schematic representation of the dorsal pancreas bud dissection from E12.5 mouse embryo. (A) Brightfield image of E12.5 mouse embryo. The upper body of the embryo (above the liver) and the tail region are removed to isolate the mid-body (B). Incisions are shown by red dotted lines. (C) Further dissection results in exposure of the stomach and duodenum region (indicated by the dotted box). The dorsal pancreatic bud (see red dotted circle) is at the base of the stomach and next to the spleen primordium. The dissected dorsal pancreatic bud (D) is transferred with a Pasteur pipette into the microwell of a glass bottom dish containing BME culture medium (E, F). Abbreviations: sto (stomach), sp (spleen), dp (dorsal pancreatic bud), duo (duodenum). Scale bars, 1000 &mu;m (A, C).
<img alt="Figure 2" src="/files/ftp_upload/3979/3979fig2.jpg" /> 
Figure 2. Whole-mount confocal immunofluorescence analysis of pancreatic explant cultures. (A) Brightfield images of pancreatic explants on day 1 and day 2 of ex vivo culture. Red dotted line indicates initiation of branching of the epithelium. (B) 3D reconstruction of whole-mount immunostaining for Pdx1 on day 3 pancreatic culture. Pancreatic explants showed tubules of Pdx1+ cells undergoing extensive branching by 48 hr of cultures. (C) Whole-mount immunostaining of day 3 pancreatic explant with antibodies against the basolateral membrane marker E-cadherin (Ecad), tip progenitor marker, Carboxypeptidase 1 (Cpa1) and phospho-Histone H3 (pHH3). Most of the mitotic active pHH3+ cells colocalize at the peripheral tips with Cpa1+ cells. Dashed lines indicate tip of the branches. (D) Whole-mount immunostaining for endocrine lineage markers, insulin (Ins) and glucagon (Gluca) at day 3. Yellow arrows indicate clusters of Ins+ and Gluca+ cells. Inset (D'), higher-magnification of one of the endocrine cell cluster. (E) Whole-mount immunostaining of day 3 pancreatic explant for Pdx1, &beta;1 integrin (&beta;1int) and F-actin (Fact). Mesenchymal cells (asterisks) interspersed among Pdx1-positive epithelial cells. Images shown in C, D and E are single confocal optical sections of Z-series. Scale bars, 100 &mu;m (A, B); 50 &mu;m (C-E).
<img alt="Figure 3" src="/files/ftp_upload/3979/3979fig3.jpg" /> 
Figure 3. Representative live-cell imaging of ex vivo cultured pancreatic explant. (A-D) Frames from a time-lapse movie of a pancreatic explant from a mT/mG mouse transgenic embryo. Time of imaging is shown in hours:minutes. The explant was imaged starting at Day 1. Z-stack images were acquired with a 40X oil immersion objective every 12 min for a total of 15 hr. Dashed lines indicate tip of the branches and border between epithelium and mesenchyme. Scale bar, 50 &mu;m.

<ol>
	<li>Slack, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+biology+of+the+pancreas.">Developmental biology of the pancreas.</a> Development. 121, 1569-1580 (1995).</li><li>Pan, F., Wright, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreas+organogenesis:+from+bud+to+plexus+to+gland.">Pancreas organogenesis: from bud to plexus to gland.</a> Dev. Dyn. 240, 530-565 (2011).</li><li>Puri, S., Hebrok, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+Plasticity+within+the+Pancreas-+Lessons+Learned+from+Development.">Cellular Plasticity within the Pancreas- Lessons Learned from Development.</a> Developmental Cell. 18, 342-356 (2010).</li><li>Spagnoli, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+endoderm+to+pancreas:+a+multistep+journey.">From endoderm to pancreas: a multistep journey.</a> Cell. Mol. Life Sci. 64, 2378-2390 (2007).</li><li>Hick, A. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+primitive+duct+formation+in+the+pancreas+and+submandibular+glands:+a+role+for+SDF-1.">Mechanism of primitive duct formation in the pancreas and submandibular glands: a role for SDF-1.</a> BMC Dev. 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No conflicts of interest declared.

Once pancreatic fate is specified, pancreatic progenitor cells undergo extensive proliferation, differentiation and morphogenesis to eventually form a mature and functional organ2,4. At present, how branching takes place in the pancreas and how it is connected to progenitor proliferation and differentiation is largely unknown. Pancreatic explant cultures represent an ideal system to elucidate these processes ex vivo5,9,11. By combining live-cell imaging with ex vivo explants...

<table border="1">
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>Antibodies: Carboxypeptidase E-cadherin F-actin Glucagon Insulin &beta;1-integrin Pdx1 Pdx1 Phospho-Histone H3</td>
 <td>AbD Serotec Invitrogen Molecular Probes ImmunoStar Millipore Millipore Abcam Hybridoma bank Cell Signalling</td>
 <td>1810-0006 13-1900 A-12373 20076 4011-01 MAB1997 ab47267 F109-D12 9706</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Basal Medium Eagle (BME)</td>
 <td>Sigma</td>
 <td>B1522-500ML</td>
 <td>Kept in sterile conditions</td>
 </tr>
 <tr>
 <td>Cell culture grade water</td>
 <td>PAA</td>
 <td>S15-012</td>
 <td>Kept in sterile conditions</td>
 </tr>
 <tr>
 <td>Culture dishes (glass-bottomed), 35-mm </td>
 <td>MatTek Corporation </td>
 <td>P35G-0-20-C </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Donkey Serum</td>
 <td>Chemicon</td>
 <td>S30-100 ml</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fetal calf serum Gold</td>
 <td>PAA</td>
 <td>A15-151</td>
 <td>Kept in sterile conditions</td>
 </tr>
 <tr>
 <td>Fibronectin</td>
 <td>Invitrogen</td>
 <td>330100-8</td>
 <td>Stock sol. 1 mg/ml in cell culture grade water</td>
 </tr>
 <tr>
 <td>Gentamicin </td>
 <td>Invitrogen</td>
 <td>15750-037</td>
 <td>Kept in sterile conditions</td>
 </tr>
 <tr>
 <td>Glutamine</td>
 <td>Invitrogen</td>
 <td>25030-024</td>
 <td>Kept in sterile conditions</td>
 </tr>
 <tr>
 <td>4-well Multidishes</td>
 <td>Nunc</td>
 <td>176740</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Microscopes: Inverted Confocal Microscope (LSM 700) Stereomicroscope (Discovery V12)</td>
 <td>Zeiss Zeiss</td>
 <td>&nbsp;</td>
 <td>Objectives: C-Apochromat 10X / 0.45 W M27 (work. dist. 1.8 mm; imaging depth ~100 mm); C-Apochromat 40X / 1.2 W Corr M27 (work. dist. 0.28 mm; ~imaging depth 50 &mu;m) Transillumination from below and fiber-optic illumination from above</td>
 </tr>
 <tr>
 <td>Paraformaldehyde</td>
 <td>Roth</td>
 <td>0335.3</td>
 <td>Stock solution 20% </td>
 </tr>
 <tr>
 <td>Pasteur Pipet (Glass), 150 mm</td>
 <td>VWR</td>
 <td>HECH567/1</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Penicillin/Streptomycin</td>
 <td>PAA</td>
 <td>P11-010</td>
 <td>Kept in sterile conditions</td>
 </tr>
 <tr>
 <td>Petri dishes, 60 mm</td>
 <td>Greiner Bio-One</td>
 <td>628102</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Petri dishes, 35 mm</td>
 <td>Greiner Bio-One</td>
 <td>627161</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>1X PBS, pH7.4</td>
 <td>PAA</td>
 <td>H15-002</td>
 <td>Kept in sterile conditions</td>
 </tr>
 <tr>
 <td>Spring Scissors 8 mm blade curved</td>
 <td>Fine Science Tools</td>
 <td>15023-10</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Triton-X100</td>
 <td>Roth</td>
 <td>3051.3</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Watchmaker's foreceps Dumont #5</td>
 <td>Roth</td>
 <td>K342.1</td>
 <td>&nbsp;</td>
 </tr>
 </table>

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.

Watch this Scientific Journal Video about A System for ex vivo Culturing of Embryonic Pancreas at JoVE.com

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.

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 ...

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13.5K Views.  Max-Delbrück-Center for Molecular Medicine. 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.

Video: A System for ex vivo Culturing of Embryonic Pancreas

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 ...

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not regenerate very efficiently in vivo; and as a result, osteoarthritis leads to irreversible cartilage loss and is accompanied by chronic pain and immobility 1,2. Cartilage tissue engineering offers promising potential to regenerate and restore tissue function. This technology typically involves seeding chondrocytes into natural or synthetic scaffolds and culturing the resulting 3D construct in a balanced medium over a period of time with a goal of engineering a biochemically and biomechanically mature tissue that can be transplanted into a defect site in vivo 3-6. Achieving an optimal condition for chondrocyte growth and matrix deposition is essential for the success of cartilage tissue engineering. 
 In the native joint cavity, cartilage at the articular surface of the bone is bathed in synovial fluid. This clear and viscous fluid provides nutrients to the avascular articular cartilage and contains growth factors, cytokines and enzymes that are important for chondrocyte metabolism 7,8. Furthermore, synovial fluid facilitates low-friction movement between cartilaginous surfaces mainly through secreting two key components, hyaluronan and lubricin 9 10. In contrast, tissue engineered cartilage is most often cultured in artificial media. While these media are likely able to provide more defined conditions for studying chondrocyte metabolism, synovial fluid most accurately reflects the natural environment of which articular chondrocytes reside in.
 Indeed, synovial fluid has the advantage of being easy to obtain and store, and can often be regularly replenished by the body. Several groups have supplemented the culture medium with synovial fluid in growing human, bovine, rabbit and dog chondrocytes, but mostly used only low levels of synovial fluid (below 20&#37;) 11-25. While chicken, horse and human chondrocytes have been cultured in the medium with higher percentage of synovial fluid, these culture systems were two-dimensional 26-28. Here we present our method of culturing human articular chondrocytes in a 3D system with a high percentage of synovial fluid (up to 100&#37;) over a period of 21 days. In doing so, we overcame a major hurdle presented by the high viscosity of the synovial fluid. This system provides the possibility of studying human chondrocytes in synovial fluid in a 3D setting, which can be further combined with two other important factors (oxygen tension and mechanical loading) 29,30 that constitute the natural environment for cartilage to mimic the natural milieu for cartilage growth. Furthermore, This system may also be used for assaying synovial fluid activity on chondrocytes and provide a platform for developing cartilage regeneration technologies and therapeutic options for arthritis.

A 3D system of culturing human articular chondrocytes in high levels of synovial fluid is described. Synovial fluid reflects the most natural microenvironment for articular cartilage, and can be easily obtained and stored. This system thus can be used for studying cartilage regeneration and for screening therapeutics for treating arthritis.

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not ...

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 ...

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 ...

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles1,2. Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture3-6. Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue3,6. High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture6. By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6&#160;cultures in parallel.

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

We describe the dissection and ex vivo culture of mouse embryonic skin. The culture system maintains an air-liquid interface across the tissue surface and allows imaging on an inverted microscope. Melanoblasts, a component of the developing skin, are fluorescently labeled allowing their behavior to be observed using confocal microscopy.

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...

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.

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.

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 ...

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells 1. The whole embryonic organ can be cultured at multiple stages of development 2-4. These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens. 
We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. 
We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell&#180;s tensegrity.

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The three-dimensional culture method described in this protocol recapitulates pancreas development from dispersed embryonic mouse pancreas progenitors, including their substantial expansion, differentiation and morphogenesis into a branched organ. This method is amenable to imaging, functional interference and manipulation of the niche.

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the ...