We thank the staff of AniCan (CRCL, Lyon) for their technical assistance with animal care. This work was supported by the Institut National de la Sant&eacute; Et de la Recherche M&eacute;dicale (INSERM Avenir Program), the Ligue Nationale Contre le Cancer, by the Association pour la Recherche sur le Cancer, by the Institut National du Cancer, and by fellowships from the Ligue Nationale Contre le Cancer (JG), from the Institut National du Cancer (JG), from the Minist&egrave;re de l'Enseignement Sup&eacute;rieur et de la Recherche of France (RMP and DFV) and from the Association pour la Recherche sur le Cancer (DFV).

All procedures were approved by an ethic committee under regulatory of governmental authority (&quot;Comit&eacute; d&#39;Evaluation Commun au Centre L&eacute;on B&eacute;rard, &agrave; l&#39;Animalerie de transit de l&#39;ENS, au PBES et au laboratoire P4&quot; (CECCAPP)). Mice were maintained in a specific pathogen-free animal facility at the &quot;Plateforme AniCan, Centre L&eacute;on B&eacute;rard&quot; (Lyon, France) and handled in compliance with the institutional guidelines.

	A schematic representation of the procedure is shown in Figure 1.

	1. Pancreas Dissection and Dilaceration (Day 0)

	A very rapid dissection is critical for an optimal yield of extraction and to insure a good viability of cells in culture. In order to reduce the time needed for pancreas isolation, all instruments and equipment must be ready before the mouse euthanasia.
<ol>
	<li>
		Euthanize mouse by CO2 asphyxiation or cervical dislocation.</li>
</ol>

	From this step, all procedures have to be performed under a sterile atmosphere (microbiological safety cabinet, level II) with sterile dissection equipment.
<ol start="2">
	<li>
		Fix the mouse and spray the mouse abdomen with 70% ethanol. With any dissecting scissors and forceps, make a V-shaped incision at the genital area and continue it up to the diaphragm to open completely the abdominal cavity.</li>
	<li>
		Position the liver lobes against the diaphragm; they should remain there if the body cavity is open far enough. Pull the gut and the colon outside the abdominal cavity to your left, and find the rectum. With a pair of curved forceps and dissecting scissors, grab and section the rectum.</li>
	<li>
		With the same pair of forceps, carefully unroll entirely the bowel from the rectum to the stomach by pulling the intestine on your left.</li>
</ol>

	At this step, the pancreas can be distinguished as a small strip between the stomach and the beginning of the bowel. Its ligations with the spleen remain intact.
<ol start="5">
	<li>
		Using Noyes scissors and a pair of forceps, carefully cut the pancreas along the bowel and liberate it with the spleen from the rest of the digestive tract.</li>
	<li>
		Grab the spleen and section the pancreas attached to it (Figure 2).</li>
</ol>

	At this step, be sure that no mesenteric fat tissue and/or other adjacent tissue (spleen, bowel, etc) could be collected with the pancreas, to avoid cellular contamination.

	For the rest of the procedure, all buffers must be prepared without calcium ion Ca2+ chelators to avoid the complete dissociation of the exocrine pancreatic tissue in single acinar cells.
<ol start="7">
	<li>
		Rinse the pancreas twice in Hank&#39;s Balanced Salt Solution (HBSS) 1x.</li>
</ol>

	At this step, as fat tissue will float contrary to pancreas that will sink, it is easily possible to visualize and rapidly remove the contaminant white adipose tissue still attached to pancreas.

	If the pancreas needs to be transported to the cell culture facility, it must be kept on ice in HBSS&nbsp;1x.
<ol start="8">
	<li>
		Transfer the pancreas in a sterile Petri dish containing 5 ml of HBSS 1x. Using Noyes scissors and a scalpel, slice the pancreas in small pieces of 1 to 3 mm3 (Figure 3A).</li>
</ol>

	2. Enzymatic and Mechanical Dissociations of Pancreas (Day 0)
<ol>
	<li>
		Transfer them into a sterile 50 ml polypropylene tube.</li>
	<li>
		Centrifuge for 2 min at 450 x g and 4 &deg;C. Aspirate and discard the supernatant to remove cell fragments and blood cells.</li>
	<li>
		Add 10 ml of collagenase IA solution (HBSS 1x containing 10 mM HEPES, 200 U/ml of collagenase IA, and 0.25 mg/ml of trypsin inhibitor) to pancreas sections. Using a 25 ml serological pipette, transfer them to a 25 cm2 flask. Incubate it for 20-30 min at 37 &deg;C. During this time (every 5 min), perform a mechanical dissociation by energetically moving back-and-forth the pancreas fragments about ten times, in sterile pipettes of decreasing size (25, 10, and 5 ml serological pipettes).</li>
</ol>

	At this step, it is essential to frequently monitor the extent of the enzymatic dissociation of pancreatic sections.
<ol start="4">
	<li>
		When the pancreatic tissue seems to be well-dissociated (according to the disappearance of pancreatic fragments and to the increased turbidity of the solution) (Figure 3B), stop the enzymatic reaction by adding 10 ml of cold buffered washing solution (HBSS 1x containing 5 % Fetal Bovine Serum (FBS) and 10 mM HEPES).</li>
	<li>
		Transfer it into a sterile 50 ml polypropylene tube and centrifuge for 2 min at 450 x g and 4 &deg;C. Carefully aspirate and discard the supernatant to remove the collagenase IA solution.</li>
	<li>
		Resuspend and wash the pellet with 10 ml of buffered washing solution. Centrifuge for 3 min at 450 x g and 4 &deg;C. Carefully aspirate and discard the supernatant. Repeat this step two more times.</li>
</ol>

	3. Filtration and Seeding of Dispersed Acini (Day 0)
<ol>
	<li>
		Resuspend the cell pellet in 7 ml of Waymouth&#39;s medium containing 2.5 % FBS, 1 % Penicillin-Streptomycin mixture (PS), 0.25 mg/ml of trypsin inhibitor, and 25 ng/ml of recombinant human Epidermal Growth Factor (EGF).</li>
	<li>
		Filtrate the cell mixture by allowing it to pass through a 100 &mu;m filter to retain the non-digested fragments (ducts, blood vessels, and Langerhans islets). Pancreatic acinar structures (acinus of 10-15 cells) pass through.</li>
	<li>
		Rinse the filter with 6 ml of Waymouth&#39;s medium containing FBS, PS, trypsin inhibitor, and EGF.</li>
</ol>

	After this step, the cells have to be treated very carefully, to avoid any acini dissociation.
<ol start="4">
	<li>
		Seed the isolated acini in a 6-well culture dish (2 ml per well) (Figure 3C). Culture them at 37 &deg;C under 5% (v/v) CO2 atmosphere.</li>
</ol>

	After this step, the acinar cells are cultured in suspension.

	4. Acinar Cell Culture (Day 1 to 10)
<ol>
	<li>
		Twenty-four hours after, transfer the acini (in suspension) into a new 6-well culture dish, to eliminate the contaminant cells and cellular remnants that have adhered overnight (Figure 4).</li>
</ol>

	If the cell culture needs to be extended for several days or if the experimental conditions require cells grown in monolayer, it is recommended to transfer and seed acini on matrix scaffolds.
<ol start="2">
	<li>
		The day before seeding on matrix support (Day 0), coat a 6-well culture dish with type I collagen (5 &mu;g/cm2). Add 1 ml of type I collagen solution (50 &mu;g/ml in 0.02 M acetic acid, 0.2 &mu;m-filtered) to each well and allow it to passively adsorb on plastic, during 1 hr at 37 &deg;C (or overnight at 4 &deg;C).</li>
	<li>
		Aspirate the type I collagen solution and rinse the coated well twice with Phosphate-Buffered Saline 1x.</li>
	<li>
		Allow the coated well to dry (under a microbiological safety cabinet) at least 12 hr before use.</li>
	<li>
		Transfer the isolated primary acini (obtained at Step 4.1) into the type I collagen-coated 6-well culture dish and culture them in the same conditions as previously described (at 37 &deg;C under 5% (v/v) CO2 atmosphere). The cells adhere to the type I collagen substrate for 2 days.</li>
	<li>
		On Day 3, change the culture medium to eliminate non-viable cells that have not adhered. With time in culture, the cells will progressively spread on the collagen-containing support. Change the culture medium every 3 days (Figure 5).</li>
</ol>

	The isolated acinar cells obtained can be counted, after a complete mechanical dissociation by using a Thoma cell counting chamber. Note that isolated acinar cells cannot be maintained in culture afterwards.

	The quality of the acinar culture obtained can be controlled by checking the expression of acinar specific markers such as Trypsinogen, Pancreas Transcription Factor 1 subunit Alpha, or Carboxypeptidase A1 (by immunocytochemistry or immunofluorescence experiments).

<ol>
	<li>Lardon, J., Bouwens, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metaplasia+in+the+pancreas.">Metaplasia in the pancreas.</a> Differentiation. 73, 278-286 (2005).</li><li>Sphyris, N., Logsdon, C. D., Harrison, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+retention+of+zymogen+granules+in+cultured+murine+pancreatic+acinar+cells+and+induction+of+acinar-ductal+transdifferentiation+in+vitro.">Improved retention of zymogen granules in cultured murine pancreatic acinar cells and induction of acinar-ductal transdifferentiation in vitro.</a> Pancreas. 30, 148-157 (2005).</li><li>Amsterdam, A., Jamieson, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+characterization+of+isolated+pancreatic+exocrine+cells.">Structural and functional characterization of isolated pancreatic exocrine cells.</a> Proc. Natl. Acad. Sci. U.S.A. 69, 3028-3032 (1972).</li><li>Amsterdam, A., Jamieson, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+dispersed+pancreatic+exocrine+cells.+I.+Dissociation+technique+and+morphologic+characteristics+of+separated+cells.">Studies on dispersed pancreatic exocrine cells. I. Dissociation technique and morphologic characteristics of separated cells.</a> J. Cell. Biol. 63, 1037-1056 (1974).</li><li>Amsterdam, A., Jamieson, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+dispersed+pancreatic+exocrine+cells.+II.+Functional+characteristics+of+separated+cells.">Studies on dispersed pancreatic exocrine cells. II. Functional characteristics of separated cells.</a> J. Cell. Biol. 63, 1057-1073 (1974).</li><li>Schultz, G. S., 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=Guinea+pig+pancreatic+acini+prepared+with+purified+collagenase.">Guinea pig pancreatic acini prepared with purified collagenase.</a> Exp. Cell Res. 130, 49-62 (1980).</li><li>Williams, J. A., Korc, M., Dormer, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Action+of+secretagogues+on+a+new+preparation+of+functionally+intact,+isolated+pancreatic+acini.">Action of secretagogues on a new preparation of functionally intact, isolated pancreatic acini.</a> Am. J. Physiol. 235, 517-524 (1978).</li><li>Logsdon, C. D., Williams, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+growth+factor+binding+and+biologic+effects+on+mouse+pancreatic+acini.">Epidermal growth factor binding and biologic effects on mouse pancreatic acini.</a> Gastroenterology. 85, 339-345 (1983).</li><li>Han, B., Logsdon, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholecystokinin+induction+of+mob-1+chemokine+expression+in+pancreatic+acinar+cells+requires+NF-kappaB+activation.">Cholecystokinin induction of mob-1 chemokine expression in pancreatic acinar cells requires NF-kappaB activation.</a> Am. J. Physiol. 277, 74-82 (1999).</li><li>Ji, B., Kopin, A. S., Logsdon, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Species+differences+between+rat+and+mouse+CCKA+receptors+determine+the+divergent+acinar+cell+response+to+the+cholecystokinin+analog+JMV-180.">Species differences between rat and mouse CCKA receptors determine the divergent acinar cell response to the cholecystokinin analog JMV-180.</a> J. Biol. Chem. 275, 19115-19120 (2000).</li><li>Ji, K. A., Yang, M. S., Jou, I., Shong, M. H., Joe, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombin+induces+expression+of+cytokine-induced+SH2+protein+(CIS)+in+rat+brain+astrocytes:+involvement+of+phospholipase+A2,+cyclooxygenase,+and+lipoxygenase.">Thrombin induces expression of cytokine-induced SH2 protein (CIS) in rat brain astrocytes: involvement of phospholipase A2, cyclooxygenase, and lipoxygenase.</a> Glia. 48, 102-111 (2004).</li><li>Gaiser, S., 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=Intracellular+activation+of+trypsinogen+in+transgenic+mice+induces+acute+but+not+chronic+pancreatitis.">Intracellular activation of trypsinogen in transgenic mice induces acute but not chronic pancreatitis.</a> Gut. 60, 1379-1388 (2011).</li><li>Logsdon, C. D., Williams, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+acinar+cells+in+monolayer+culture:+direct+trophic+effects+of+caerulein+in+vitro.">Pancreatic acinar cells in monolayer culture: direct trophic effects of caerulein in vitro.</a> Am. J. Physiol. 250, 440-447 (1986).</li><li>Stanger, B. Z., Dor, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+cellular+origins+of+pancreatic+cancer.">Dissecting the cellular origins of pancreatic cancer.</a> Cell Cycle. 5, 43-46 (2006).</li><li>Vincent, D. F., 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=Tif1gamma+suppresses+murine+pancreatic+tumoral+transformation+by+a+smad4-independent+pathway.">Tif1gamma suppresses murine pancreatic tumoral transformation by a smad4-independent pathway.</a> Am. J. Pathol. 180, 2214-2221 (2012).</li><li>Vincent, D. F., 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=Inactivation+of+TIF1gamma+cooperates+with+Kras+to+induce+cystic+tumors+of+the+pancreas.">Inactivation of TIF1gamma cooperates with Kras to induce cystic tumors of the pancreas.</a> PLoS Genet. 5, e1000575 (2009).</li><li>Dorrell, 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=Isolation+of+mouse+pancreatic+alpha,+beta,+duct+and+acinar+populations+with+cell+surface+markers.">Isolation of mouse pancreatic alpha, beta, duct and acinar populations with cell surface markers.</a> Mol. Cell Endocrinol. 339, 144-150 (2011).</li><li>Case, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis,+intracellular+transport+and+discharge+of+exportable+proteins+in+the+pancreatic+acinar+cell+and+other+cells.">Synthesis, intracellular transport and discharge of exportable proteins in the pancreatic acinar cell and other cells.</a> Biol. Rev. Camb. Philos. Soc. 53, 211-354 (1978).</li><li>Blauer, M., Nordback, I., Sand, J., Laukkarinen, J. <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+explant+outgrowth+culture+model+for+mouse+pancreatic+acinar+cells+with+long-term+maintenance+of+secretory+phenotype.">A novel explant outgrowth culture model for mouse pancreatic acinar cells with long-term maintenance of secretory phenotype.</a> Eur. J. Cell. Biol. 90, 1052-1060 (2011).</li></ol>

The authors declare that they have no competing financial interests.

In this protocol, we describe a procedure for isolating pancreatic acinar cells. This method makes possible to isolate more than 20 x 106 acinar cells per animal in less than 1 hr. Thanks to its rapid and simple implementation (as many as 10 pancreases can be independently processed per experiment in parallel), this protocol appears as a good compromise between existing isolation methods 3-5,9-12,17 .

	Critical steps/Trouble-shooting
<p class="jove_content...

A frequently encountered problem for research laboratories working on exocrine pancreatic tissue is the difficulty of cultivating acinar cells in vitro for a period of time long enough to allow a long-term experiment.
One factor impeding development of such culture systems is the intrinsic sensitivity of pancreatic tissue to experimental manipulation due to the high content in glycolytic, proteolytic, and lipolytic enzymes, which literally digest the pancreatic tissue when they are released during the isolation of pancreatic cells.
A second factor is the remarkable in vitro plasticity of acinar cells, which tend to lose their secretory characteristics and transdifferentiate to other mature cells, such as pancreatic ductal cells or hepatocyte-like cells 1. In vitro, this cell plasticity varies with the experimental conditions (such as culture medium composition) 2 and introduces a degree of complexity into the design of appropriate culture conditions for exocrine pancreatic cells 1.
Several methods have been developed for the isolation and culture of acinar cells, first from the guinea pig pancreas 3-5. Initially, those protocols involved digestion of pancreatic tissue with collagenase, chymotrypsin, and a protease cocktail, with ultimate isolation by vigorous mechanical dissociation. The pancreatic cells isolated in this way displayed abnormal structural and functional characteristics, notably a loss of apical structures and significant damage to their membrane receptors. Isolated cells remained viable for only 1 or 2 days.
Preparation of dispersed acini maintains their intra- and intercellular architecture, preserving cell membranes, limiting damage to surface receptors, and thus improving exocrine secretion in response to secretagogues 6-8. As a result, this method offers the major advantage of extending acinar cell viability to 7-10 days in vitro. Furthermore, this method is currently preferred to acinar cell isolation 9-12 because maintenance of intercellular contacts, including cell coupling by gap junctions, is an essential determinant of the exocrine pancreatic acinar cell phenotype 13.
As the dedifferentiation of acinar cells and their transdifferentiation to ductal cells is one of the proposed mechanisms for the genesis of aggressive exocrine pancreatic cancers 14, the dispersed acini model is also an adequate system to study pancreatic plasticity and its subsequent molecular mechanisms. Furthermore, in combination with the use of genetically modified animals 15,16 and the development of gene transfer techniques (adenoviral 2 or lentiviral transduction, use of nanoparticles, etc), this in vitro primary acinar cell model can be very useful in determining how various genetic dysfunctions affect the regulation of acinar cell differentiation or dedifferentiation and should provide better understanding of the molecular events responsible for the onset of pancreatitis, precancerous lesions, and changes in cell plasticity.
Isolation of dispersed acini is the approach we use in our laboratory to culture pancreatic acinar cells. We here describe and discuss the method used. It involves enzymatic dissociation of pancreatic tissue (with a bacterial collagenase) coupled to mechanical disruption without dissociation of acinar cells. While most protocols involve culturing the acini, either in suspension or on specially treated plastic substrates, we grow them in suspension only briefly (for 24 hr), seeding them afterwards onto matrix scaffolds if prolonged cell culture is required.
This protocol allows rapid isolation (in less than 1 hr) of dispersed pancreatic acini, sustainable for more than one week in culture. It allows isolation of more than 20 x 106 acinar cells per mouse pancreas. Its simplicity makes it possible to process independently as many as 10 pancreases in parallel. By maintaining the intra- and intercellular architecture of acini and thus the acinar phenotype of isolated primary cells, this model constitutes a system of choice for the study of transdifferentiation mechanisms, as all other exocrine pancreatic models currently available are derived from pancreatic tumors displaying many genetic alterations leading to cellular transformation.

<table><tbody><tr><td>0.2 &amp;mu;m filter</td><td>Dutscher</td><td>146560</td><td></td></tr><tr><td>10 ml serological pipettes</td><td>Beckton Dickinson</td><td>357551</td><td></td></tr><tr><td>100 &amp;mu;m filter</td><td>Beckton Dickinson</td><td>352360</td><td></td></tr><tr><td>100 mm Petri dish</td><td>Beckton Dickinson</td><td>353003</td><td></td></tr><tr><td>1000 &amp;mu;l filter tips</td><td>Starlab</td><td>S1122-1830</td><td></td></tr><tr><td>20 &amp;mu;l filter tips</td><td>Starlab</td><td>S1120-1810</td><td></td></tr><tr><td>200 &amp;mu;l filter tips</td><td>Starlab</td><td>S1120-8810</td><td></td></tr><tr><td>25 ml serological pipettes</td><td>Beckton Dickinson</td><td>357535</td><td></td></tr><tr><td>5 ml serological pipettes</td><td>Beckton Dickinson</td><td>357543</td><td></td></tr><tr><td>50 ml polypropylene tube</td><td>Beckton Dickinson</td><td>352070</td><td></td></tr><tr><td>6-well plate</td><td>Beckton Dickinson</td><td>353046</td><td></td></tr><tr><td>Acetic acid 100%</td><td>VWR BDH Prolabo</td><td>20104.298</td><td></td></tr><tr><td>Collagenase IA</td><td>Sigma-Aldrich</td><td>C2676</td><td></td></tr><tr><td>Curved forceps, Dumont #7</td><td>World Precision Instruments</td><td>14188</td><td>To sterilize before use</td></tr><tr><td>Dissecting scissors, straight</td><td>World Precision Instruments</td><td>14393</td><td>To sterilize before use</td></tr><tr><td>Epidermal Growth Factor, human</td><td>Promokine</td><td>C-60180</td><td></td></tr><tr><td>Ethanol absolute (AnalaR Normapur)</td><td>VWR BDH Prolabo</td><td>20821.310</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Lonza</td><td>14-801F</td><td></td></tr><tr><td>Forceps, Dumont #5</td><td>World Precision Instruments</td><td>14098</td><td>To sterilize before use</td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution 1x </td><td>Gibco</td><td>14025050</td><td></td></tr><tr><td>HEPES 1 M (pH 6.98-7.30)</td><td>Lonza</td><td>17-737F</td><td></td></tr><tr><td>Incubator O&lt;sub&gt;2&lt;/sub&gt;/CO&lt;sub&gt;2&lt;/sub&gt;</td><td>Sanyo</td><td>MCO-19M</td><td></td></tr><tr><td>Inverted microscope</td><td>Nikon</td><td>Eclipse TS100</td><td></td></tr><tr><td>Matrigel</td><td>Beckton Dickinson</td><td>356234</td><td></td></tr><tr><td>Microbiological Safety Cabinet, level II</td><td>Faster</td><td>SafeFast Elite 212 S</td><td></td></tr><tr><td>Noyes scissors, sharp/sharp tips, German</td><td>World Precision Instruments</td><td>500228-G</td><td>To sterilize before use</td></tr><tr><td>Penicillin-Streptomycin mixture</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Phosphate Buffer Saline 10x </td><td>Gibco</td><td>14200067</td><td></td></tr><tr><td>Pipet-Aid</td><td>Drummond Scientific Company</td><td>Pipet-Aid XP</td><td></td></tr><tr><td>Pipetman P1000</td><td>Gilson</td><td>F123602</td><td></td></tr><tr><td>Pipetman P20</td><td>Gilson</td><td>F123600</td><td></td></tr><tr><td>Pipetman P200</td><td>Gilson</td><td>F123601</td><td></td></tr><tr><td>Refrigerated centrifuge</td><td>Eppendorf</td><td>5810R</td><td></td></tr><tr><td>Scalpel</td><td>Paramount Surgimed Ltd.</td><td>Disposable Scalpel Size 23</td><td></td></tr><tr><td>T25 flask, 25 cm&lt;sup&gt;2&lt;/sup&gt;</td><td>Sigma-Aldrich</td><td>Z707481</td><td></td></tr><tr><td>Trypsin inhibitor, from &lt;em&gt;Glycine Max&lt;/em&gt;</td><td>Sigma-Aldrich</td><td>T6522</td><td></td></tr><tr><td>Type I collagen</td><td>Beckton Dickinson</td><td>354236</td><td></td></tr><tr><td>Waymouth&amp;rsquo;s medium</td><td>Gibco</td><td>31220-023</td><td></td></tr></tbody></table>

isolation and culture of mouse primary pancreatic acinar cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

Figure 1 schematizes the "dispersed" acini method for primary acinar cells isolation. The critical steps, which have to be strictly respected during the protocol, are described in the discussion part.

	To facilitate its removal, the pancreas has to be collected from the abdomen along with the attached spleen (Figure 2). Both organs need to be cut apart, and the residual fat tissue that could be still attached to the pancreas must be removed (Step 1.6).
...

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Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

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35.5K Views.  Centre de Recherche en Cancérologie de Lyon. In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

Video: Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture

The differentiation of acinar cells to ductal cells during pancreatitis and in the early development of pancreatic cancer is a key process that requires further study. To understand the mechanisms regulating acinar-to-ductal metaplasia (ADM), ex vivo 3D culture and differentiation of primary acinar cells to ductal cells offers many advantages over other systems. With the technique herein, modulation of protein expression is simple and quick, requiring only one day to isolate, stimulate or virally infect, and begin culturing primary acinar cells to investigate the ADM process. In contrast to using basement membrane matrix, the seeding of acinar cell clusters in collagen I extracellular matrix, allows acinar cells to retain their acinar identity before manipulation. This is vital when testing the contribution of various components to the induction of ADM. Not only are the effects of cytokines or other ectopically administered factors testable through this technique, but the contribution of common mutations, increased protein expression, or knockdown of protein expression is testable via viral infection of primary acinar cells, using adenoviral or lentiviral vectors. Moreover, cells can be re-isolated from collagen or basement membrane matrix at the endpoint and analyzed for protein expression.

Primary acinar cell isolation, protein expression or activity modulation, culture, and down-stream applications are abundantly useful in the ex vivo study of acinar-to-ductal metaplasia (ADM), an early event in the development of pancreatic cancer.

The differentiation of acinar cells to ductal cells during pancreatitis and in the early development of pancreatic cancer is a key process that requires ...

Cancer Research

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

Medicine

A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic &#946; cells which will lead to the development of type 2 diabetes mellitus (T2DM). Although the mechanisms for the chemical-induced &#946; cell damage were unclear and likely to be complex, one recurring finding is that these chemicals induce oxidative stress leading to the generation of excessive reactive oxygen species (ROS) which induce damage to the &#946; cell. To identify potential diabetogenic environmental chemicals, we isolated pancreatic islet cells from C57BL/6 mice and cultured islet cells in 96-well cell culture plates; then, the islet cells were dosed with chemicals and the ROS generation was detected by 2',7'-dichlorofluorescein (DCFH-DA) fluorescent dye. Using this method, we found that bisphenol A (BPA), Benzo[a]pyrene (BaP), and polychlorinated biphenyls (PCBs), could induce high levels of ROS, suggesting that they may potentially induce damage in islet cells. This method should be useful for screening diabetogenic xenobiotics. In addition, the cultured islet cells may also be adapted for in vitro analysis of chemical-induced toxicity in pancreatic cells.

Here we present a protocol to isolate mouse pancreatic islet cells for screening the ROS inductions by the xenobiotics in order to identify the potential diabetogenic xenobiotic chemicals.

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic &#946; cells which will lead to ...

Immunology and Infection

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

Preparation of Pancreatic Acinar Cells for the Purpose of Calcium Imaging, Cell Injury Measurements, and Adenoviral Infection

The pancreatic acinar cell is the main parenchymal cell of the exocrine pancreas and plays a primary role in the secretion of pancreatic enzymes into the pancreatic duct. It is also the site for the initiation of pancreatitis. Here we describe how acinar cells are isolated from whole pancreas tissue and intracellular calcium signals are measured. In addition, we describe the techniques of transfecting these cells with adenoviral constructs, and subsequently measuring the leakage of lactate dehydrogenase, a marker of cell injury, during conditions that induce acinar cell injury in vitro. These techniques provide a powerful tool to characterize acinar cell physiology and pathology.

We describe a reproducible method of preparing mouse pancreatic acinar cells from a mouse for the purpose of examining acinar cell calcium signals and cellular injury with physiologically and pathologically relevant stimuli. A method for adenoviral infection of these cells is also provided.

The pancreatic acinar  cell is the main parenchymal cell of the exocrine pancreas and plays a primary  role in the secretion of pancreatic enzymes into ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

De Novo Generation of Somatic Stem Cells by YAP/TAZ

Here we present protocols to isolate primary differentiated cells and turn them into stem/progenitor cells (SCs) of the same lineage by transient expression of the transcription factor YAP. With this method, luminal differentiated (LD) cells of the mouse mammary gland are converted into cells that exhibit molecular and functional properties of mammary SCs. YAP also turns fully differentiated pancreatic exocrine cells into pancreatic duct-like progenitors. Similarly, to endogenous, natural SCs, YAP-induced stem-like cells ("ySCs") can be eventually expanded as organoid cultures long term in vitro, without further need of ectopic YAP/TAZ, as ySCs are endowed with a heritable self-renewing SC-like state. 
The reprogramming procedure presented here offers the possibility to generate and expand in vitro progenitor cells of various tissue sources starting from differentiated cells. The straightforward expansion of somatic cells ex vivo has implications for regenerative medicine, for understanding mechanisms of tumor initiation and, more in general, for cell and developmental biology studies.

De Novo Generation of Somatic Stem Cells by YAP/TAZ

Availability of somatic SCs is crucial for regenerative medicine, disease modeling and to gain insight into SC properties. Here we present experimental strategies to reprogram, in vitro, differentiated adult cells into their corresponding expandable tissue-specific stem/progenitor cells by the transient expression of the single transcriptional co-activator YAP.

Here we present protocols to isolate primary differentiated cells and turn them into stem/progenitor cells (SCs) of the same lineage by transient ...

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from &#946;-cells), glucagon (&#945;-cells) and somatostatin (&#948;-cells). &#946;-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, &#945;-cells and &#948;-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest.
Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of &#946;-cells is minimized.
Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase.
Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells ...

Streamlining Mouse Pancreatic Endothelial Cell Extraction for Disease Studies

Isolation of Mouse Pancreatic Endothelial Cells

The pancreas is a vital organ for maintaining metabolic balance within the body, in part due to its production of metabolic hormones such as insulin and glucagon, as well as digestive enzymes. The pancreas is also a highly vascularized organ, a feature facilitated by the intricate network of pancreatic capillaries. This extensive capillary network is made up of highly fenestrated endothelial cells (ECs) important for pancreas development and function. Accordingly, the dysfunction of ECs can contribute to that of the pancreas in diseases like diabetes and cancer. Thus, researching the function of pancreatic ECs (pECs) is important not only for understanding pancreas biology but also for developing its pathologies. Mouse models are valuable tools to study metabolic and cardiovascular diseases. However, there has not been an established protocol with sufficient details described for the isolation of mouse pECs due to the relatively small population of ECs and the abundant digestive enzymes potentially released from the acinar tissue that can lead to cell damage and, thus, low yield. To address these challenges, we devised a protocol to enrich and recover mouse pECs, combining gentle physical and chemical dissociation and antibody-mediated selection. The protocol presented here provides a robust method to extract intact and viable ECs from the whole mouse pancreas. This protocol is suitable for multiple downstream assays and may be applied to various mouse models.

This protocol describes the isolation of mouse endothelial cells from whole pancreas.

The pancreas is a vital organ for maintaining metabolic balance within the body, in part due to its production of metabolic hormones such as insulin and ...

Acinar-to-Ductal Metaplasia Induction: A Method to Study Acinar Cell to Ductal Cell Differentiation in 3D Ex Vivo Culture

Source: Fleming Martinez, A. K. et. al. <a href="https://www.jove.com/v/59096/" target="_blank">Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture</a>. J. Vis. Exp. (2019).
This video describes the technique of differentiating pancreatic acinar cells to ductal cells in ex vivo 3D culture. This helps to understand the mechanisms regulating the acinar-to-ductal metaplasia (ADM), a key process in the early development of pancreatic cancer.

Source: Fleming Martinez, A. K. et. al. Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture. J. Vis. Exp. ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

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Chapters in this video

Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture

Lepr^db Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Preparation of Pancreatic Acinar Cells for the Purpose of Calcium Imaging, Cell Injury Measurements, and Adenoviral Infection

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

De Novo Generation of Somatic Stem Cells by YAP/TAZ

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Isolation of Mouse Pancreatic Endothelial Cells

Acinar-to-Ductal Metaplasia Induction: A Method to Study Acinar Cell to Ductal Cell Differentiation in 3D Ex Vivo Culture