This work was funded sequentially by a NCCR Frontiers in Genetics pilot award, Juvenile Diabetes Research Foundation Grant 41-2009-775 and Grant 12-126875 from Det Frie Forskningsr&#229;d/Sundhed og Sygdom. The authors thank the Spagnoli lab for hosting the video shooting.

This protocol aims to grow pancreatic organoids derived from murine E10.5 dissociated epithelial pancreatic cells.
The protocol requires ethical approval for animal experimentation.
1. Dissection of Dorsal Pancreatic Bud from E10.5 Mouse Embryos
<ol>
	<li>Sacrifice timed-pregnant mice at embryonic day (E) 10.5, open the abdomen with a pair of scissors, remove the two uterine horns and place them in a 10 cm Petri dish filled with cold phosphate buffer saline (PBS) or Dulbecco modified essential medium (DMEM) kept on ice. The total experiment from the sacrifice to cell seeding is done in 60-90 min to prevent cell damage.</li>
	<li>Separate the uterus into individual embryo segments using small scissors. Transfer one embryo to a 35 mm Petri dish with cold PBS and visualize it under a dissecting microscope with illumination from above. With dissection forceps remove the surrounding muscle, decidua and yolk sac and expose the embryo. Place the embryo back into cold DMEM medium. Embryos can easily be transferred using a 3 ml plastic transfer pipette/dropper. Isolate every individual embryo in a similar manner before continuing.</li>
	<li>Place an embryo into a clean 35 mm Petri dish in PBS.
	<ol>
		<li>Use thin forceps (0.05 mm width) to remove the forelimb. Gently insert the forceps in the opening to detach the digestive tract from the spinal cord region (Figure 1). OPTIONAL: To more conveniently see the stomach, remove the upper body of the embryo, down to the heart and the tail region below the yolk stalk.</li>
		<li>Locate the stomach, liver and intestine. Using the forceps, isolate the gastrointestinal tract from the stomach to the intestine and place it in cold DMEM on ice (Figure 1). The dorsal pancreatic bud is attached dorsally, posterior to the stomach (Figure 1).</li>
		<li>Isolate every individual gastrointestinal tract in a similar manner before continuing. If the embryos need to be genotyped, collect the tail at the time of dissection and keep every individual gastrointestinal tract in a different well in a 24-well plate with cold DMEM on ice.</li>
	</ol>
	</li>
	<li>Place one gastrointestinal tract in a clean 35 mm Petri dish in cold PBS. Now use illumination from below in bright field to visualize and dissect the dorsal pancreatic bud under the dissection microscope. These conditions will optimize the visualization of volumes. Using electrolytically-sharpened tungsten needles or 20 G syringe needles, isolate the pancreatic bud with as little mesenchyme as possible around the epithelium (Figure 1).
	<ol>
		<li>Transfer the isolated bud into a petri dish containing a cold dispase solution (1.25 mg/ml) for 2-3 min. From this point, transfer can most conveniently be done with flame-pulled 50 &#181;l glass capillaries attached to a mouth-controlled flexible tube. Alternatively, but with more risk of losing the bud, use a 10 &#181;l automatic pipette (Pipetman) with appropriate plastic tips.</li>
		<li>Perform pancreatic bud aspiration and ejection under microscopic control. Put the pancreatic bud back in PBS. Further clean the isolated pancreatic bud from the mesenchyme with the needles and gentle aspiration using the glass capillary (Figure 1).</li>
		<li>When the entire mesenchyme is removed, rinse the pancreatic bud in cold PBS; transfer each bud to cold DMEM in individual wells (60-well mini-trays filled with 10 &#181;l of cold DMEM). It is important not to remove the mesenchyme in dispase, which makes the tissue very sticky.</li>
	</ol>
	</li>
</ol>
2. Plating and Culture of Dispersed Cells
<ol>
	<li>Transfer the dissected epithelia from all embryos with a flame-pulled glass capillary into conical wells of 60-well mini-trays filled with 10 &#181;l PBS for rinsing.
	<ol>
		<li>Transfer the bud into 10 &#181;l of Trypsin 0.05 % and let it incubate at 37 &#176;C for 4 min. Inactivate the trypsin by transferring the bud into a well with 10 &#181;l DMEM + 10% fetal calf serum (FCS).</li>
		<li>Dissociate the cell suspension by aspiration through a thin capillary pulled with a pipette puller. It is important to avoid bubbles at this stage while pipetting up and down to dissociate the cells. Pancreas organoids optimally start from small groups of 5-15 cells and therefore partial dissociation is recommended (Figure 1).</li>
	</ol>
	</li>
	<li>Pool the cells from several embryos into an Eppendorf tube in order to minimize differences due to individual processing. Dilute the cell suspension in chilled Matrigel at a 1:3 ratio. Aliquot this mixture to a 96-well plate, 8 &#181;l/well or in a plate optimized for imaging (see below).</li>
	<li>Incubate the plate at 37 &#176;C for 5 min, allowing the Matrigel to thicken. Fill the wells with 70 &#181;l of medium of choice (organoid or sphere, see Tables 1 and 2) and leave in a humidified environment containing 5% CO2 and 95% air at 37 &#176;C.</li>
	<li>Replace the medium every 4th day. Monitor the growing pancreatic colonies daily and document the process by imaging.</li>
	<li>Small molecules or proteins of interest can be added to the medium at this stage for interference experiments, as reported previously 28.</li>
</ol>
Table 1: Organoid medium.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Name of Material</td>
			<td>Stock Concentration</td>
			<td>Concentration in final medium</td>
			<td>Volume of stock</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>100%</td>
			<td>1%</td>
			<td>50 &#181;l</td>
		</tr>
		<tr>
			<td>KnockOut Serum replacement (supplement)</td>
			<td>100%</td>
			<td>10%</td>
			<td>500 &#181;l</td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>14.3 M</td>
			<td>0.1 mM</td>
			<td>1 &#181;l</td>
		</tr>
		<tr>
			<td>Phorbol Myristate Acetate (PMA)</td>
			<td>16 &#181;M</td>
			<td>16 nM</td>
			<td>5 &#181;l</td>
		</tr>
		<tr>
			<td>Y-27632 (ROCK inhibitor)</td>
			<td>50 mM</td>
			<td>10 &#181;M</td>
			<td>1 &#181;l</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>50 &#181;g/ml</td>
			<td>25 ng/ml</td>
			<td>2.5 &#181;l</td>
		</tr>
		<tr>
			<td>Recombinant Human R-spondin 1</td>
			<td>250 &#181;g/ml</td>
			<td>500 ng/ml</td>
			<td>10 &#181;l</td>
		</tr>
		<tr>
			<td>&#160;- or -&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse R-spondin 1</td>
			<td>250 &#181;g/ml</td>
			<td>500 ng/ml</td>
			<td>10 &#181;l</td>
		</tr>
		<tr>
			<td>Recombinant Human FGF1 (aFGF)</td>
			<td>100 &#181;g/ml</td>
			<td>25 &#181;g/ml</td>
			<td>1.25 &#181;l</td>
		</tr>
		<tr>
			<td>Heparin (Liquemin)</td>
			<td>2500 U/ml</td>
			<td>2.5 U/ml</td>
			<td>2 &#181;l</td>
		</tr>
		<tr>
			<td>Recombinant Human FGF10</td>
			<td>100 &#181;g/ml</td>
			<td>100 ng/ml</td>
			<td>5 &#181;l</td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td></td>
			<td></td>
			<td>4,412.25 &#181;l</td>
		</tr>
		<tr>
			<td>Total</td>
			<td></td>
			<td></td>
			<td>5,000 &#181;l</td>
		</tr>
	</tbody>
</table>
Table 2: Sphere medium.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Name of Material</td>
			<td>Stock Concentration</td>
			<td>Concentration in final medium</td>
			<td>Volume of stock</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>100%</td>
			<td>1%</td>
			<td>50 &#181;l</td>
		</tr>
		<tr>
			<td>B27 x50 (supplement)</td>
			<td>100%</td>
			<td>10%</td>
			<td>100 &#181;l</td>
		</tr>
		<tr>
			<td>Recombinant Human FGF2 (bFGF)</td>
			<td>100 &#181;g/ml</td>
			<td>64 ng/ml</td>
			<td>3.2 &#181;l</td>
		</tr>
		<tr>
			<td>Y-27632 (ROCK inhibitor)</td>
			<td>50 mM</td>
			<td>10 &#181;M</td>
			<td>1 &#181;l</td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td></td>
			<td></td>
			<td>4845.8 &#181;l</td>
		</tr>
		<tr>
			<td>Total</td>
			<td></td>
			<td></td>
			<td>5000 &#181;l</td>
		</tr>
	</tbody>
</table>
3. Imaging of the Progression of Organoid Development
<ol>
	<li>Image organoids either daily or by time lapse microscopy using a fluorescent time-lapse microscope. For time lapse imaging, use an XY(Z) automated inverted fluorescent microscope.</li>
	<li>Deposit small droplets of 3 &#181;l in 4-well plates or glass-bottom plates filled with 2-5 ml medium. Image with a 10x long distance objective. Note: Transgenic mice expressing fluorescent tracers can be used. Movie 1 shows for example the initial expansion of organoids from Pdx1-Ngn3-ERTM-ires-nGFP+ mice4. The nuclear GFP enables the user to track cells as individual objects but similar principles can be applied to track cells with membrane fluorescence such as mT/mG mice29.</li>
	<li>Start time-lapse imaging 3 hr after seeding the cells to avoid focus drifts and set the software controlling automation to take 1 picture/hr for 3 or more days at manually defined positions. For every position, acquire a differential interference contrast (DIC) image as well as the GFP signal, reporting fluorescent marker expression.</li>
</ol>
4. Recovery of Organoids for Histology
<ol>
	<li>Place the 96-well plate on ice and remove the medium, replacing it with ice cold PBS. This partially depolymerizes Matrigel.</li>
	<li>Gently aspirate each individual organoid, removing the surrounding Matrigel using a 1,000 &#181;l tip in order to not disrupt the overall architecture. Transfer each organoid to a well with ice cold PBS. Keep the plate on ice. Direct fixation in Matrigel is also possible.</li>
	<li>Fix the organoid for 15 min in 4% paraformaldehyde (PFA), cryopreserve it in sucrose and embed it in gelatin. Process each organoid for cryosectioning and histology as previously described (Johansson et al., 2007) 4.</li>
</ol>
5. Recovery of Organoids for PCR and Biochemistry
<ol>
	<li>Place the 96-well plate on ice and remove the medium. Add 60 &#181;l of RNAlater per well in order to stabilize and protect cellular RNA.</li>
	<li>Disrupt the gel in each well mechanically by partially depolymerizing it on ice. Either recover individual organoids using a 1,000 &#181;l tip in order to not disrupt the overall architecture or recover the entire well (with Matrigel) by disrupting the gel mechanically with a 200 &#181;l tip. Use a 1,000 &#181;l tip to transfer the well content into an RNAse free-non-sticky Eppendorf tube kept on ice.</li>
	<li>Wash the wells with 60 &#181;l RNAlater and add the remaining content to the same Eppendorf tube.</li>
	<li>Spin the tubes for 5 min at 500-1,000 x g at 4 &#176;C.</li>
	<li>Remove the supernatant, only leaving 20-30 &#181;l of RNAlater in the tube together with the pellet. For biochemistry, store the samples as dry as possible.</li>
	<li>Do not freeze samples in RNAlater immediately; store at 4 &#176;C O/N (to allow RNAlater to thoroughly penetrate the tissue). The tissue can be stored at -20 &#176;C for long term storage and can later be processed for tissue disruption and extraction of small quantities of RNA.</li>
</ol>

<ol>
	<li>Pagliuca, F. W., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+make+a+functional+beta-cell.">How to make a functional beta-cell.</a> Development. 140, 2472-2483 (2013).</li><li>Percival, A. C., Slack, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+pancreatic+development+using+a+cell+lineage+label.">Analysis of pancreatic development using a cell lineage label.</a> Exp Cell Res. 247, 123-132 (1999).</li><li>Attali, 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=Control+of+beta-cell+differentiation+by+the+pancreatic+mesenchyme.">Control of beta-cell differentiation by the pancreatic mesenchyme.</a> Diabetes. 56, 1248-1258 (2007).</li><li>Johansson, K. A., 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=Temporal+control+of+neurogenin3+activity+in+pancreas+progenitors+reveals+competence+windows+for+the+generation+of+different+endocrine+cell+types.">Temporal control of neurogenin3 activity in pancreas progenitors reveals competence windows for the generation of different endocrine cell types.</a> Dev Cell. 12, 457-465 (2007).</li><li>Speier, S., Rupnik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+approach+to+in+situ+characterization+of+pancreatic+beta-cells.">A novel approach to in situ characterization of pancreatic beta-cells.</a> Pflugers Arch. 446, 553-558 (2003).</li><li>Golosow, N., Grobstein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epitheliomesenchymal+interaction+in+pancreatic+morphogenesis.">Epitheliomesenchymal interaction in pancreatic morphogenesis.</a> Dev Biol. 4, 242-255 (1962).</li><li>Miralles, F., Czernichow, P., Scharfmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Follistatin+regulates+the+relative+proportions+of+endocrine+versus+exocrine+tissue+during+pancreatic+development.">Follistatin regulates the relative proportions of endocrine versus exocrine tissue during pancreatic development.</a> Development. 125, 1017-1024 (1998).</li><li>Petzold, K. M., 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=A+system+for+ex+vivo+culturing+of+embryonic+pancreas.">A system for ex vivo culturing of embryonic pancreas.</a> J. Vis. Exp. , 3979 (2012).</li><li>Miralles, F., Battelino, T., Czernichow, P., Scharfmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta+plays+a+key+role+in+morphogenesis+of+the+pancreatic+islets+of+Langerhans+by+controlling+the+activity+of+the+matrix+metalloproteinase+MMP-2.">TGF-beta plays a key role in morphogenesis of the pancreatic islets of Langerhans by controlling the activity of the matrix metalloproteinase MMP-2.</a> J Cell Biol. 143, 827-836 (1998).</li><li>Hope, K., Bhatia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+interrogation+of+stem+cells.">Clonal interrogation of stem cells.</a> Nat Methods. 8, 36-40 (2011).</li><li>Sugiyama, T., Rodriguez, R. T., McLean, G. W., Kim, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+markers+of+fetal+pancreatic+epithelium+permit+prospective+isolation+of+islet+progenitor+cells+by+FACS.">Conserved markers of fetal pancreatic epithelium permit prospective isolation of islet progenitor cells by FACS.</a> Proc Natl Acad Sci U S A. 104, 175-180 (2007).</li><li>Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+neurons+and+astrocytes+from+isolated+cells+of+the+adult+mammalian+central+nervous+system.">Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.</a> Science. 255, 1707-1710 (1992).</li><li>Dontu, G., 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=In+vitro+propagation+and+transcriptional+profiling+of+human+mammary+stem/progenitor+cells.">In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells.</a> Genes Dev. 17, 1253-1270 (2003).</li><li>Smukler, S. R., 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=The+adult+mouse+and+human+pancreas+contain+rare+multipotent+stem+cells+that+express+insulin.">The adult mouse and human pancreas contain rare multipotent stem cells that express insulin.</a> Cell Stem Cell. 8, 281-293 (2011).</li><li>Seaberg, R. 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=Clonal+identification+of+multipotent+precursors+from+adult+mouse+pancreas+that+generate+neural+and+pancreatic+lineages.">Clonal identification of multipotent precursors from adult mouse pancreas that generate neural and pancreatic lineages.</a> Nat Biotechnol. 22, 1115-1124 (2004).</li><li>Jin, L., 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=Colony-forming+cells+in+the+adult+mouse+pancreas+are+expandable+in+Matrigel+and+form+endocrine/acinar+colonies+in+laminin+hydrogel.">Colony-forming cells in the adult mouse pancreas are expandable in Matrigel and form endocrine/acinar colonies in laminin hydrogel.</a> Proc Natl Acad Sci U S A. 110, 3907-3912 (2013).</li><li>Sugiyama, 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=Reconstituting+pancreas+development+from+purified+progenitor+cells+reveals+genes+essential+for+islet+differentiation.">Reconstituting pancreas development from purified progenitor cells reveals genes essential for islet differentiation.</a> Proc Natl Acad Sci U S A. 110, 12691-12696 (2013).</li><li>Huch, 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=Unlimited+in+vitro+expansion+of+adult+bi-potent+pancreas+progenitors+through+the+Lgr5/R-spondin+axis.">Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis.</a> Embo J. , (2013).</li><li>Sato, 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=Single+Lgr5+stem+cells+build+crypt-villus+structures+in+vitro+without+a+mesenchymal+niche.">Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.</a> Nature. , (2009).</li><li>Ootani, A., 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=Sustained+in+vitro+intestinal+epithelial+culture+within+a+Wnt-dependent+stem+cell+niche.">Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche.</a> Nat Med. 15, 701-706 (2009).</li><li>Barker, N., 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=Lgr5(+ve)+stem+cells+drive+self-renewal+in+the+stomach+and+build+long-lived+gastric+units+in+vitro.">Lgr5(+ve) stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro.</a> Cell Stem Cell. 6, 25-36 (2010).</li><li>Huch, 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=In+vitro+expansion+of+single+Lgr5++liver+stem+cells+induced+by+Wnt-driven+regeneration.">In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration.</a> Nature. 494, 247-250 (2013).</li><li>Lukacs, R. U., Goldstein, A. S., Lawson, D. A., Cheng, D., Witte, O. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+cultivation+and+characterization+of+adult+murine+prostate+stem+cells.">Isolation, cultivation and characterization of adult murine prostate stem cells.</a> Nat Protoc. 5, 702-713 (2010).</li><li>Rock, J. R., 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=Basal+cells+as+stem+cells+of+the+mouse+trachea+and+human+airway+epithelium.">Basal cells as stem cells of the mouse trachea and human airway epithelium.</a> Proc Natl Acad Sci U S A. 106, 12771-12775 (2009).</li><li>Eiraku, 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=Self-organizing+optic-cup+morphogenesis+in+three-dimensional+culture.">Self-organizing optic-cup morphogenesis in three-dimensional culture.</a> Nature. 472, 51-56 (2011).</li><li>Spence, J. R., 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=Directed+differentiation+of+human+pluripotent+stem+cells+into+intestinal+tissue+in+vitro.">Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro.</a> Nature. 470, 105-109 (2011).</li><li>Lancaster, M. A., 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=Cerebral+organoids+model+human+brain+development+and+microcephaly.">Cerebral organoids model human brain development and microcephaly.</a> Nature. 10, (2013).</li><li>Greggio, C., 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=Artificial+three-dimensional+niches+deconstruct+pancreas+development+in+vitro.">Artificial three-dimensional niches deconstruct pancreas development in vitro.</a> Development. 140, 4452-4462 (2013).</li><li>Muzumdar, M. D., Tasic, B., Miyamichi, K., Li, L., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+global+double-fluorescent+Cre+reporter+mouse.">A global double-fluorescent Cre reporter mouse.</a> Genesis. 45, 593-605 (2007).</li></ol>

The authors have nothing to disclose.

Large-scale production of functional beta cells in vitro is still ineffective 1. In this challenging context, developmental biology studies may help deciphering the exact signals that are required for the differentiation of functional beta cells. This protocol allows for the maintenance, expansion and differentiation of embryonic pancreatic progenitors in vitro. This includes the formation of insulin-producing beta cells that do not co-express other endocrine hormones, have high levels of Pdx...

Organ culture provides a useful model that bridges the gaps between the complex but highly relevant in vivo investigations and the convenient but approximate simulation of cell line models. In the case of the pancreas, there is no cell line perfectly equivalent to pancreas progenitors although there are transformed cell lines simulating endocrine and exocrine cells. The adult whole pancreas cannot be cultured; isolated endocrine islets can be maintained for few weeks without cell proliferation and tissue slices can be kept in vitro for few hours 5. Embryonic pancreas culture has been widely used not only to study its development, but also to investigate epithelial-mesenchymal interactions 4,6,7, to image processes 8 or to chemically interfere with them 9. Two organ culture methods are mainly used: the first consists in culturing pancreatic buds on fibronectin coated plates 2, which is convenient for imaging purposes; the second option is to culture the organs on filters at the air-liquid interface 3,4 which best preserves morphogenesis. Although very useful, these methods lead to a certain degree of flattening; the expansion of progenitors is very limited as compared to the normal development and the starting population is complex comprising all types of pancreatic cells and mesenchymal cells.
The ability to culture and expand dispersed primary cells is valuable to study lineage relationships and uncover the intrinsic properties of isolated cell types 10. Sugiyama et al. 11 could maintain pancreas progenitors and endocrine progenitors that retained some functional characters for 3-5 days in culture on feeder layers. Pancreatospheres, akin to neurospheres 12 and mammospheres 13, have been expanded from adult islets and ductal cells although the nature of the progenitors/stem cells that generate these spheres is not clear. In addition, in contrast with physiological development, the pancreatospheres contained some neurons 14,15. Spheres were also recently produced from embryonic pancreas progenitors 16,17 and regenerating pancreata18 with good progenitor expansion and subsequent differentiation but failed to recapitulate morphogenesis.
3D models from dispersed and often defined cells that self-organize into miniaturized organs have recently flourished and simulate the development or adult turnover of multiple organs such as the intestine 19,20, the stomach 21, the liver 22, the prostate 23 and the trachea 24. In some instances, developmental morphogenesis and differentiation have been recapitulated in 3D from ES cells, as is the case of optic cups 25, intestine 26 or brain 27.
Here, we describe a method to expand dissociated multipotent pancreatic progenitors in a 3D Matrigel scaffold where they can differentiate and self-organize.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>KnockOut Serum replacement (supplement)</td>
			<td>Gibco</td>
			<td>10828-028</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Sigma Aldrich</td>
			<td>3148-25ML</td>
			<td>Stock keept at 4&#176;C</td>
		</tr>
		<tr>
			<td>Phorbol Myristate Acetate (PMA)</td>
			<td>Calbiotech</td>
			<td>524400-1MG</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>Y-27632 (ROCK inhibitor)</td>
			<td>Sigma Aldrich</td>
			<td>ab120129</td>
			<td>Stock keept at -20&#176;C- Attention! Stability/source is a frequent source of problems</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Sigma Aldrich</td>
			<td>E9644-2MG</td>
			<td>Stock keept at -80&#176;C</td>
		</tr>
		<tr>
			<td>Recombinant Human R-spondin 1</td>
			<td>R&#38;D</td>
			<td>4645-RS-025/CF</td>
			<td>Stock keept at -80&#176;C</td>
		</tr>
		<tr>
			<td>&#160;- or -&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse R-spondin 1</td>
			<td>R&#38;D</td>
			<td>3474-RS-050</td>
			<td>Stock keept at -80&#176;C</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human FGF1 (aFGF)</td>
			<td>R&#38;D</td>
			<td>232-FA-025</td>
			<td>Stock keept at -80&#176;C- do not include to increase beta cell production</td>
		</tr>
		<tr>
			<td>Heparin (Liquemin)</td>
			<td>Drossapharm</td>
			<td></td>
			<td>Stock keept at 4&#176;C</td>
		</tr>
		<tr>
			<td>Recombinant Human FGF10</td>
			<td>R&#38;D</td>
			<td>345-FG-025</td>
			<td>Stock keept at -80&#176;C</td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>Gibco</td>
			<td>21331-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>B27 x50 (supplement)</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>Recombinant Human FGF2 (bFGF)</td>
			<td>R&#38;D</td>
			<td>233-FB-025</td>
			<td>Stock keept at -80&#176;C</td>
		</tr>
		<tr>
			<td>Y-27632 (ROCK inhibitor)</td>
			<td>Sigma Aldrich</td>
			<td>ab120129</td>
			<td>Stock keept at -20&#176;C- Attention! Stability/source is a frequent source of problems</td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>Gibco</td>
			<td>21331-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>356231</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>Trypsin 0.05%</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td>Stock keept at 4&#176;C</td>
		</tr>
		<tr>
			<td>RNAlater - RNA stabilizing reagent</td>
			<td>Qiagen</td>
			<td>76104</td>
			<td>Store at room temperature</td>
		</tr>
		<tr>
			<td>Dispase&#160;</td>
			<td>Sigma Aldrich</td>
			<td>D4818-2MG</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>BSA for reconstitution</td>
			<td>Milipore</td>
			<td><a href="http://www.millipore.com/catalogue/item/81-068-3">81-068</a></td>
			<td>For reconstituition of cytokines&#160; - Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>Fetal calf serum (FCS)</td>
			<td>Gibco</td>
			<td>16141079</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>60 well MicroWell trays</td>
			<td>Sigma Aldrich</td>
			<td>M0815-100EA</td>
			<td></td>
		</tr>
		<tr>
			<td>4-well plates</td>
			<td>Thermo Scientific</td>
			<td>176740</td>
			<td></td>
		</tr>
		<tr>
			<td>95-well plates F bottom</td>
			<td>Greiner Bio</td>
			<td>6555180</td>
			<td></td>
		</tr>
		<tr>
			<td>Glas bottom plates</td>
			<td>Ibidi</td>
			<td>81158</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposal micropittes</td>
			<td>Blaubrand</td>
			<td>708745</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

E10.5 dorsal pancreatic progenitors dissociated and seeded in 3D Matrigel recapitulate pancreas development. Progenitors can be most easily followed with fluorescent reporters. In our case we used a transgenic mouse that expresses a nuclear GFP protein controlled by Pdx1 promoter (Pdx1-Ngn3-ERTM-nGFP) (Movie 1) in the absence of tamoxifen and thus without activating Neurog3 4 (Figure 2).
With the organoid medium, an ini...

Watch this Scientific Journal Video about In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors at JoVE.com

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.

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 ...

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Biology

13.7K Views.  Swiss Institute for Experimental Cancer Research. 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.