This work was supported by an R01 grant (CA200572) from the NIH to PS. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health.The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

All animal work was approved by the Mayo Clinic IACUC.
1. Preparation of Materials, Solutions, and 3-dimensional Matrix Bases
<ol>
	<li>Cut 500 &#181;m and 105 &#181;m polypropylene meshes into 76 mm by 76 mm squares. Fold each square in half twice to create a smaller folded square. Place one 500 &#181;m and one 105 &#181;m mesh square into an autoclavable pouch.</li>
	<li>Autoclave polypropylene mesh squares, as well as two pairs of scissors and forceps.</li>
	<li>Make 100 mL of 10x Waymouth&#39;s solution. Stir 1 vial (14 g) of Waymouth&#39;s powder into 50 mL of deionized (DI) water and, when dissolved, add 30 mL of 7.5% (75 g/L) sodium bicarbonate. Bring the final volume to 100 mL using DI water and filter sterilize.
	<ol>
		<li>Store 10x Waymouth&#39;s media at 4 &#176;C and use to prepare collagen gels and 1x Waymouth&#39;s media. When precipitates form, discard.</li>
	</ol>
	</li>
	<li>Make 50 mL of 1x Waymouth&#39;s complete media per pancreas by adding 125 &#181;L of 40 mg/mL soybean trypsin inhibitor, 12.5 &#181;L of 4 mg/mL dexamethasone, and 500 &#181;L of fetal bovine serum (FBS). Sterilize by filtration and use within 48 h.</li>
	<li>Prepare 3-dimensional matrix bases using either collagen or basement membrane matrix (see Table of Materials for product information). When pipetting the base, place the plate on ice, avoid bubbles, and rotate the plate immediately after pipetting each well&#39;s volume to ensure full coverage.
	<ol>
		<li>Create collagen bases by mixing the following components on ice in a tissue culture hood: 5.5 mL of type I rat tail collagen, 550 &#181;L of 10x Waymouth&#39;s Media, and 366.6 &#181;L of 0.34 M NaOH (filtered). 
		NOTE: This is enough solution to make collagen bases for one 24-well, 12-well, or 6-well plate. One plate is sufficient for acinar isolation from one pancreas.</li>
		<li>Use the following volumes to plate the collagen base: 80 &#181;L (8-well glass slide), 200 &#181;L (24-well plate), 400 &#181;L (12-well plate), or 800 &#181;L (6-well plate).</li>
		<li>For the basement membrane matrix bases, pipet the matrix without any additional components. Use the following volumes for each well: 50 &#181;L (8-well glass slide), 120 &#181;L (24-well plate), 240 &#181;L (12-well plate), or 600 &#181;L (6-well plate).</li>
		<li>Let the bases solidify for at least 30 min in a cell culture incubator (37 &#176;C, 5% CO2) before creating a second layer of matrix with embedded cells on top of these bases (step 4).</li>
	</ol>
	</li>
	<li>In preparation for acinar isolation keep one 600 mL beaker, three 50 mL tubes, an autoclaved pair of scissors, an autoclaved pair of forceps, and an ice bucket under the hood with three weigh boats. Then, make 30 mL of HBSS with 300 &#181;L of 100x penicillin-streptomycin, putting 10 mL into each of two weigh boats and keeping the final 10 mL in a 50 mL tube (for use in steps 1.8.2 and 2.1.4).</li>
	<li>Make 40 mL of HBSS + 5% FBS, 20 mL of HBSS + 30% FBS, and 5 mL of 2 mg/mL collagenase in HBSS. Sterilize the collagenase by filtration (0.22 &#181;m pore) and keep at room temperature. Keep each of the HBSS solutions on ice.</li>
	<li>Assemble a workspace for pancreas dissection, which can be done on a lab bench.
	<ol>
		<li>Place an absorbent pad on the table, along with a polystyrene lid covered in foil. Then place a paper towel with 4 pins on top of the foil.</li>
		<li>Keep the following items within reach of the dissection workspace: an incineration bag, one set of autoclaved scissors and forceps, a spray bottle with 70% ethanol, and an ice bucket (with the 50 mL tube of HBSS containing penicillin-streptomycin from step 1.6).</li>
	</ol>
	</li>
	<li>Set a centrifuge to 4 &#176;C and a shaker to 37 &#176;C.</li>
</ol>
2. Acinar Cell Isolation
<ol>
	<li>Sacrifice the mouse via CO2 induction, and perform cervical dislocation. Immediately dissect the pancreas. To dissect the pancreas, first pin the paws of the mouse to the polystyrene lid, orient the mouse such that the tail is facing the researcher, and spray the abdomen with 70% ethanol. 
	NOTE: The mouse used in the representative results was a 12-week old non-transgenic female with C57BL/6 background. Mouse selection is further discussed in the discussion section.
	<ol>
		<li>Using a set of autoclaved scissors and forceps, lift the fur/skin with the forceps at the midline and use the scissors to make an incision through the fur and skin from the urethral opening to the diaphragm/ribcage area.</li>
		<li>Make additional incisions to the left and right such that the fur/skin is cut away to create a clear view of the abdominal cavity. Then, cut into the peritoneal lining (down the middle and to the right and left) and pull it away from the organs, as was done with the fur/skin.</li>
		<li>Lift the intestines with the forceps and put it to the left side of the mouse creating space to see the pancreas, which is light pink in color, and attached to the spleen, which is a dark red oval. The pancreatic tissue is distinguished by its soft, spongy texture. Cut out the pancreas which will run along the stomach and intertwine with the intestines.</li>
		<li>Separate the spleen from the pancreas. Then, put the pancreas in a 50 mL tube containing 10 mL of HBSS with 1x penicillin-streptomycin and bring this to the laminar flow hood. 
		NOTE: The remaining steps of the protocol should be done utilizing sterile technique in a laminar flow hood.</li>
	</ol>
	</li>
	<li>Pour the pancreas and HBSS (with penicillin-streptomycin) into an empty weigh boat and, using forceps, wash the pancreas by swirling it. Then, move the pancreas with the forceps to a second weigh boat containing HBSS (with penicillin-streptomycin). Again, wash the pancreas by swirling.</li>
	<li>Move the pancreas to the third HBSS (with penicillin-streptomycin)-containing weigh boat and begin cutting the pancreas into small pieces of 5 mm or less. Next, pour the pancreas pieces and HBSS into an empty 50 mL tube.
	<ol>
		<li>To do this, use the forceps to move the pancreas pieces into the liquid as the weigh boat is being tipped. Pour once all the pieces are no longer attached to the weigh boat. Pick up any remaining pieces with the forceps and wash the forceps in the 50 mL tube containing the pancreas in HBSS with penicillin-streptomycin.</li>
	</ol>
	</li>
	<li>Centrifuge at 931 x g for 2 min at 4 &#176;C and then remove the HBSS (and any fat that is floating) by pipetting it off with a 5 mL pipette.</li>
	<li>Add 5 mL of collagenase (diluted in HBSS in step 1.7) to the pancreas. Ensure a sealed lid by wrapping the 50 mL tube in plastic paraffin film and then place it in an incubator shaking at 220 rpm for 20 min at 37 &#176;C. 
	NOTE: The collagenase digestion time may vary, as noted in the discussion. At the end of incubation, no large pieces of tissue should remain.</li>
	<li>To stop the dissociation, place the pancreas-containing tube on ice and add 5 mL of cold HBSS + 5% FBS. Centrifuge at 931 x g for 2 min at 4 &#176;C and then pipet off the supernatant using a 5 mL pipet.</li>
	<li>Resuspend in 10 mL of HBSS + 5% FBS and centrifuge at 931 x g for 2 min at 4 &#176;C. Pipet off the supernatant using a 5 mL pipet and repeat this step with another 10 mL of HBSS + 5% FBS.</li>
	<li>Resuspend in 5 mL of HBSS + 5% FBS and, using a P1000, transfer 1 mL at a time through a 500 &#181;m mesh into a 50 mL tube. Add an additional 5 mL of HBSS + 5% FBS through the mesh with a P1000 to wash any remaining pancreatic cells through the mesh.</li>
	<li>Put the cell suspension from the 500 &#181;m mesh through the 105 &#181;m mesh by pipetting 1 mL at a time using a P1000. Next, gently pipet the cell suspension into a tube containing HBSS + 30% FBS. A layer of cell suspension will form at the top; upon centrifugation, the acinar cells will sink to form a pellet.</li>
	<li>Centrifuge at 233 x g for 2 min at 4 &#176;C. Remove the supernatant and resuspend the pellet in 1x Waymouth&#39;s complete media.
	<ol>
		<li>If proceeding directly to embedment of cells in collagen or basement membrane matrix, resuspend in a volume of 7 mL per pancreas. If proceeding to adenoviral or lentiviral infection, resuspend in 4 mL per pancreas.</li>
	</ol>
	</li>
</ol>
3. Viral Infection
<ol>
	<li>As one pancreas is sufficient for two viral infections (control and experimental, e.g., null and cre), split the cell suspension between the lids of two 35 mm plates. Using the lid of the plates allows for infection in suspension and therefore easy movement onto the final substrate later.
	<ol>
		<li>When working with virus, soak all used pipette tips in 10% bleach for at least 15 minutes. 
		NOTE: Utilization of the 35 mm plates and the 4 mL volume works well for cells from non-transgenic mice between 8 and 16 weeks old. The plate size and volume may need to be adjusted for animals with smaller or larger pancreata.</li>
	</ol>
	</li>
	<li>For adenoviral infection, add the virus (with a titer of at least 107 TU/mL) at a 1:1,000 dilution to the lid of each plate and swirl (Figure 2, GFP adenovirus). Place the plates in a cell culture incubator (37 &#176;C, 5% CO2) and swirl every 15 minutes for 1 h. Continue incubation for an additional 2 h and then proceed to embedment of cells in collagen or basement membrane matrix.</li>
	<li>After completing viral work in the hood, soak all components in the hood with 10% bleach for at least 15 minutes and then thoroughly clean the bleach off using 70% ethanol.</li>
</ol>
4. Embedment of cells in Collagen or Basement Membrane Matrix
<ol>
	<li>Make collagen gel on ice by combining 7 mL type I rat tail collagen (3.3 mg/mL), 700 &#181;L of 10x Waymouth&#39;s media, and 466.6 &#181;L of 0.34 M NaOH.</li>
	<li>Taking equal volumes of collagen gel and cell suspension, gently mix. If adding a stimulus or inhibitor, combine this with the collagen-cell suspension. Plate one well at a time (plates from step 1.5.3); keeping the plate on ice, swirl to evenly distribute the collagen-cell layer.
	<ol>
		<li>In each well, pipet the following volumes of collagen-cell suspension: 200 &#181;L (8-well glass slide), 500 &#181;L (24-well plate), 1000 &#181;L (12-well plate), or 2000 &#181;L (6-well plate). Note that ADM is induced via stimulation with TGF-&#945; (50 ng/mL) in&#160;Figure 2.</li>
	</ol>
	</li>
	<li>For embedment of cells in basement membrane matrix, mix one-part basement membrane matrix with two-parts cell suspension. As with collagen, keep the matrix-cell suspension as well as the plate on ice. Pipet one well at a time using the same volumes noted in 4.2.1 and swirl for even distribution.</li>
	<li>Place the plate in a cell culture incubator (37 &#176;C, 5% CO2) for 30 min to solidify and then add 1x Waymouth&#39;s complete media with any stimulus or inhibitor (Figure 2 uses TGF-&#945; at 50 ng/mL). Change the media (with stimulus or inhibitor) the next day and then every other day. Depending on the stimulus, ADM can be observed between day 3 and day 5.</li>
</ol>
5. Harvesting Cells from Collagen or Basement Membrane Matrix
<ol>
	<li>Collect the following materials needed for harvesting cells from collagen: 30 mL of 1 mg/mL collagenase diluted in HBSS, 40 mL of cold HBSS, 31 mL of cold PBS, two spatulas, two 50 mL tubes, and ice. Adjust the number of spatulas, tubes and amount of solutions if comparing more than two culture conditions (where each condition is &#189; of a plate). Set the centrifuge to 4 &#176;C and set the shaking incubator to 37 &#176;C.</li>
	<li>Remove the media and use spatulas to remove the collagen disks with embedded cells. For each condition, put the disks into a separate, empty 50 mL tube.</li>
	<li>Add 10 mL of 1 mg/mL collagenase in HBSS to each 50 mL tube. Wrap the tubes with plastic paraffin film and put in a 37 &#176;C shaking incubator at 225 rpm for up to 45 minutes. Monitor the digestion and remove tubes when there is no more visible collagen.</li>
	<li>Centrifuge at 233 x g at 4 &#176;C for 2 min with minimum deceleration. Take off the supernatant and resuspend in 5 mL collagenase solution per tube. Digest in a 37 &#176;C shaking incubator at 225 rpm for 10 min or until there is no visible collagen.</li>
	<li>Stop the digestion by adding 5 mL of cold HBSS and subsequently centrifuging at 233 x g for 2 min at 4 &#176;C with minimum deceleration.
	<ol>
		<li>Resuspend in 15 mL of cold HBSS and again centrifuge at 233 x g for 2 min at 4 &#176;C with minimum deceleration. Repeat.</li>
	</ol>
	</li>
	<li>Resuspend the pellet in 1 mL of PBS and move to 1.5 mL tube. Centrifuge in a swing bucket at 233 x g for 2 min at 4 &#176;C with minimum deceleration. Remove supernatant and snap-freeze in liquid nitrogen or on dry ice. 
	NOTE: The cell pellet can be stored at -70 &#176;C or used immediately in down-stream applications.</li>
	<li>To harvest basement membrane matrix-embedded cells, pipet media from each well into a 50 mL tube on ice. Then, add basement membrane matrix recovery solution to each well and scrape the cell-gel mixture into the 50 mL tube. 
	NOTE: The volume of basement membrane matrix recovery solution to add to each well is as follows: 150 &#181;L (8-well glass slide), 420 &#181;L (24-well plate), 845 &#181;L (12-well plate), and 2,000 &#181;L (6-well plate).</li>
	<li>Rinse each well twice with the basement membrane matrix recovery solution, adding the rinses to the 50 mL tube. 
	NOTE: The volume of basement membrane matrix recovery solution for each rinse is as follows: 150 &#181;L (8-well glass slide), 420 &#181;L (24-well plate), 845 &#181;L (12-well plate), and 2,000 &#181;L (6-well plate).</li>
	<li>Leave the tube on ice for 1 h or until the basement membrane matrix dissolves and follow with centrifugation at 300 x g for 5 min at 4 &#176;C. Remove the supernatant and resuspend the pellet in 1 mL of cold PBS (may transfer to 1.5 mL tube if cell pellet is desired).</li>
	<li>Centrifuge at 3,500 x g for 3 min at 4 &#176;C. Remove the supernatant and either snap freeze the cell pellet or add lysis buffer and directly proceed to downstream applications.</li>
</ol>

<ol>
	<li>Rooman, I., Real, F. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+ductal+adenocarcinoma+and+acinar+cells:+a+matter+of+differentiation+and+development.">Pancreatic ductal adenocarcinoma and acinar cells: a matter of differentiation and development.</a> Gut. 61 (3), 449-458 (2012).</li><li>Stanger, B. Z., 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=Control+of+cell+identity+in+pancreas+development+and+regeneration.">Control of cell identity in pancreas development and regeneration.</a> Gastroenterology. 144 (6), 1170-1179 (2013).</li><li>Caldas, C., Kern, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=K-ras+mutation+and+pancreatic+adenocarcinoma.">K-ras mutation and pancreatic adenocarcinoma.</a> International Journal of Pancreatology. 18 (1), 1-6 (1995).</li><li>Kopp, J. 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=Identification+of+Sox9-dependent+acinar-to-ductal+reprogramming+as+the+principal+mechanism+for+initiation+of+pancreatic+ductal+adenocarcinoma.">Identification of Sox9-dependent acinar-to-ductal reprogramming as the principal mechanism for initiation of pancreatic ductal adenocarcinoma.</a> Cancer Cell. 22 (6), 737-750 (2012).</li><li>Morris, J. P. t., Cano, D. A., Sekine, S., Wang, S. C., 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=Beta-catenin+blocks+Kras-dependent+reprogramming+of+acini+into+pancreatic+cancer+precursor+lesions+in+mice.">Beta-catenin blocks Kras-dependent reprogramming of acini into pancreatic cancer precursor lesions in mice.</a> Journal of Clinical Investigation. 120 (2), 508-520 (2010).</li><li>Grippo, P. J., Nowlin, P. S., Demeure, M. J., Longnecker, D. S., Sandgren, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preinvasive+pancreatic+neoplasia+of+ductal+phenotype+induced+by+acinar+cell+targeting+of+mutant+Kras+in+transgenic+mice.">Preinvasive pancreatic neoplasia of ductal phenotype induced by acinar cell targeting of mutant Kras in transgenic mice.</a> Cancer Research. 63 (9), 2016-2019 (2003).</li><li>Sawey, E. T., Johnson, J. A., Crawford, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+metalloproteinase+7+controls+pancreatic+acinar+cell+transdifferentiation+by+activating+the+Notch+signaling+pathway.">Matrix metalloproteinase 7 controls pancreatic acinar cell transdifferentiation by activating the Notch signaling pathway.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (49), 19327-19332 (2007).</li><li>Liou, G. Y., 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=Macrophage-secreted+cytokines+drive+pancreatic+acinar-to-ductal+metaplasia+through+NF-kappaB+and+MMPs.">Macrophage-secreted cytokines drive pancreatic acinar-to-ductal metaplasia through NF-kappaB and MMPs.</a> Journal of Cell Biology. 202 (3), 563-577 (2013).</li><li>De La, O. J., 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=Notch+and+Kras+reprogram+pancreatic+acinar+cells+to+ductal+intraepithelial+neoplasia.">Notch and Kras reprogram pancreatic acinar cells to ductal intraepithelial neoplasia.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (48), 18907-18912 (2008).</li><li>Habbe, 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=Spontaneous+induction+of+murine+pancreatic+intraepithelial+neoplasia+(mPanIN)+by+acinar+cell+targeting+of+oncogenic+Kras+in+adult+mice.">Spontaneous induction of murine pancreatic intraepithelial neoplasia (mPanIN) by acinar cell targeting of oncogenic Kras in adult mice.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (48), 18913-18918 (2008).</li><li>Liou, G. Y., 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=Mutant+KRas-Induced+Mitochondrial+Oxidative+Stress+in+Acinar+Cells+Upregulates+EGFR+Signaling+to+Drive+Formation+of+Pancreatic+Precancerous+Lesions.">Mutant KRas-Induced Mitochondrial Oxidative Stress in Acinar Cells Upregulates EGFR Signaling to Drive Formation of Pancreatic Precancerous Lesions.</a> Cell Report. 14 (10), 2325-2336 (2016).</li><li>Hughes, P., Marshall, D., Reid, Y., Parkes, H., Gelber, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+costs+of+using+unauthenticated,+over-passaged+cell+lines:+how+much+more+data+do+we+need.">The costs of using unauthenticated, over-passaged cell lines: how much more data do we need.</a> Biotechniques. 43 (5), 577-578 (2007).</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> European Journal of Cell Biology. 90 (12), 1052-1060 (2011).</li><li>Gout, J., 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+and+culture+of+mouse+primary+pancreatic+acinar+cells.">Isolation and culture of mouse primary pancreatic acinar cells.</a> Journal of Visualized Experiments. (78), (2013).</li><li>Artym, V. V., Matsumoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+cells+in+three-dimensional+collagen+matrix.">Imaging cells in three-dimensional collagen matrix.</a> Current Protocols in Cell Biology. , 11-20 (2010).</li><li>Geraldo, S., Simon, A., Vignjevic, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revealing+the+cytoskeletal+organization+of+invasive+cancer+cells+in+3D.">Revealing the cytoskeletal organization of invasive cancer cells in 3D.</a> Journal of Visualized Experiments. (80), e50763 (2013).</li></ol>

The authors declare that they have no competing financial interests.

The relatively short amount of time for isolation, infection, and plating primary acinar cells is an advantage of this method. In contrast, culturing acinar cells from an explant outgrowth requires little hands-on time, but it takes seven days for the outgrowth of acinar cells13. An alternative protocol for acinar isolation14 notes a very short method for obtaining acinar cells; however, EGF is an essential component in keeping acinar cells alive when isolated using that pr...

Acinar-to-ductal metaplasia (ADM) is a protective mechanism during pancreatitis and a key process driving pancreatic cancer development1 that requires further mechanistic insight. While inflammation-induced ADM is reversible2, oncogenic KRAS mutations, which are present in 90% of pancreatic cancer cases3, prevent differentiation back to an acinar phenotype4,5,6. Culturing and differentiating primary acinar cells into ductal cells in 3D culture allows for study of the molecular mechanisms regulating the ADM process, which is difficult to study in vivo. Such ex vivo studies enable real-time visualization of ADM, its drivers and its regulators. Several drivers of the ADM process, as well as mechanistic insight on their downstream signaling pathways, have been identified or verified using the here described method. These include ADM induction by TGF-&#945; (transforming growth factor alpha)-mediated expression of MMP-7 (matrix metalloproteinase-7) and activation of Notch7, as well as RANTES (regulated on activation, normal T cell expressed and secreted; also known as chemokine ligand 5 or CCL5) and TNF&#945; (tumor necrosis factor alpha)-induced ADM through activation of NF-&#954;B (nuclear factor-&#954;B)8. An additional mediator of ADM is oncogenic KRas9,10, which causes a rise in oxidative stress that enhances the ADM process through increased expression of EGFR (epidermal growth factor receptor) and its ligands, EGF (epidermal growth factor) and TGF-&#945;11.
While use of pancreatic cancer cell lines is common for in vitro studies, primary cell cultures offer many advantages. For instance, primary acinar cells from non-transgenic mice or mice harboring initiating mutations, such as KrasG12D, are the most appropriate model for studying the early event of ADM because the cells have few and controlled mutations, which are known to be present early in pancreatic cancer development. This is in contrast to many pancreatic cancer cell lines which have multiple mutations and varied expression based on passage number12. Additionally, the signaling pathways identified by such means have been verified by animal studies. One caveat of using primary acinar cells is that they cannot be transfected as typical cancer cell lines can.
The method herein details the techniques of acinar cell isolation, 3D culture that mimics ADM by adding stimulants or modulating protein expression via adenoviral or lentiviral infection, as well as re-isolation of cells at the endpoint for further analyses.

<table><tbody><tr><td>37 &amp;deg;C shaking incubator</td><td>Thermo Scientific</td><td>SHKE4000-7</td><td></td></tr><tr><td>5% CO&lt;sub&gt;2&lt;/sub&gt;, 37 &amp;deg;C Incubator</td><td>NUAIRE</td><td>NU-5500</td><td></td></tr><tr><td>50 mL tubes</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>Absorbent pad, 20&quot; x 24&quot;</td><td>Fisherbrand</td><td>1420662</td><td></td></tr><tr><td>Adenovirus, Ad-GFP</td><td>Vector Biolabs&amp;nbsp;</td><td>1060</td><td></td></tr><tr><td>Aluminum foil</td><td>Fisherbrand</td><td>01-213-101</td><td></td></tr><tr><td>BD Precision Glide Needle 21 G x 1/2</td><td>Fisher Scientific&amp;nbsp;</td><td>305167</td><td>Use as pins for dissection&amp;nbsp;</td></tr><tr><td>Beaker, 600 mL</td><td>Fisherbrand</td><td>FB-101-600</td><td></td></tr><tr><td>Bleach, 8.25%</td><td>Clorox</td><td>31009</td><td></td></tr><tr><td>Cell Recovery Solution</td><td>Corning</td><td>354253</td><td>Referred to as &#039;basement membrane matrix recovery solution&#039; in the manuscript.</td></tr><tr><td>Centrifuge&amp;nbsp;</td><td>Beckman Coulter</td><td>Allegra X-15R Centrifuge</td><td></td></tr><tr><td>Collagenase Type I, Clostridium histolyticum</td><td>Sigma</td><td>C0130-1G</td><td>Create a 100 mg/mL solution by dissolving powder in sterile molecular biology grade water. When thawing one aliquot, dilute to 10 mg/ml, filter sterilize and place 1 ml aliquots at -20 &amp;deg;C.</td></tr><tr><td>Dexamethasone</td><td>Sigma</td><td>D1756</td><td>Create a 4 mg/mL solution by dissolving powder in methanol, aliquoting and storing at -20 &amp;deg;C.&amp;nbsp;</td></tr><tr><td>Ethanol, 200 proof</td><td>Decon Laboratories&amp;nbsp;</td><td>2701</td><td></td></tr><tr><td>Fetal Bovine Serum&amp;nbsp;</td><td>Sigma</td><td>F0926-100mL</td><td></td></tr><tr><td>Forceps&amp;nbsp;</td><td>Fine Science Tools</td><td>11002-12</td><td></td></tr><tr><td>Forceps&amp;nbsp;</td><td>Fine Science Tools</td><td>91127-12</td><td></td></tr><tr><td>Glass slide, 8-well</td><td>Lab-Tek</td><td>177402</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution (HBSS), No calcium, No magnesium, No phenol red</td><td>Fisher Scientific</td><td>SH3048801</td><td>&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Ice bucket, rectangular</td><td>Fisher Scientific&amp;nbsp;</td><td>07-210-103</td><td></td></tr><tr><td>Instant Sealing Sterilization Pouch</td><td>Fisherbrand</td><td>01-812-51</td><td></td></tr><tr><td>LAB GUARD specimen bags (for mouse after dissection)&amp;nbsp;</td><td>Minigrip</td><td>SBL2X69S</td><td></td></tr><tr><td>Lentiviral Packaging Mix, Virapower</td><td>Invitrogen</td><td>44-2050</td><td></td></tr><tr><td>Matrigel</td><td>Corning</td><td>356234</td><td>Referred to as &#039;basement membrane matrix&#039; in the manuscript.</td></tr><tr><td>Parafilm</td><td>Bemis</td><td>PM992</td><td>Referred to as &#039;plastic paraffin film&#039; in the manuscript.</td></tr><tr><td>PBS</td><td>Fisher Scientific</td><td>SH30028.02</td><td></td></tr><tr><td>Penicillin-Streptomycin&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>15140122</td><td></td></tr><tr><td>Pipet tips, 10 &amp;micro;L</td><td>USA Scientific</td><td>1110-3700</td><td></td></tr><tr><td>Pipet tips, 1,000 &amp;micro;L&amp;nbsp;</td><td>Olympus Plastics</td><td>24-165RL</td><td></td></tr><tr><td>Pipet tips, 200 &amp;micro;L&amp;nbsp;</td><td>USA Scientific</td><td>1111-1700</td><td></td></tr><tr><td>Pipet-Aid</td><td>Drummond</td><td></td><td></td></tr><tr><td>PIPETMAN Classic P10, 1-10 &amp;mu;L</td><td>Gilson</td><td>F144802</td><td></td></tr><tr><td>PIPETMAN Classic P1000, 200-1000 &amp;mu;L</td><td>Gilson</td><td>F123602</td><td></td></tr><tr><td>PIPETMAN Classic P20, 2-20 &amp;mu;L</td><td>Gilson</td><td>F123600</td><td></td></tr><tr><td>PIPETMAN Classic P200, 20-200 &amp;mu;L</td><td>Gilson</td><td>F123601</td><td></td></tr><tr><td>Pipettes, 10 mL&amp;nbsp;</td><td>Falcon</td><td>357551</td><td></td></tr><tr><td>Pipettes, 25 mL</td><td>Falcon</td><td>357525</td><td></td></tr><tr><td>Pipettes, 5 mL&amp;nbsp;</td><td>Falcon</td><td>357543</td><td></td></tr><tr><td>Plate, 12-well</td><td>Corning Costar</td><td>3513</td><td></td></tr><tr><td>Plate, 24-well plate</td><td>Corning Costar</td><td>3524</td><td></td></tr><tr><td>Plate, 35 mm</td><td>Falcon</td><td>353001</td><td></td></tr><tr><td>Plate, 6-well</td><td>Falcon</td><td>353046</td><td></td></tr><tr><td>Polybrene</td><td>EMD Millipore</td><td>TR-1003-G</td><td>Create a 40 mg/mL solution by dissolving powder in sterile molecular biology grade water, aliquoting and storing at -20 &amp;deg;C.&amp;nbsp;</td></tr><tr><td>Polypropylene Mesh, 105 &amp;micro;m</td><td>Spectrum Labs</td><td>146436</td><td></td></tr><tr><td>Polypropylene Mesh, 500 &amp;micro;m&amp;nbsp;</td><td>Spectrum Labs</td><td>146418</td><td></td></tr><tr><td>Scissors&amp;nbsp;</td><td>Fine Science Tools</td><td>14568-12</td><td></td></tr><tr><td>Scissors&amp;nbsp;</td><td>Fine Science Tools</td><td>91460-11</td><td></td></tr><tr><td>Sodium Bicarbonate (Fine White Powder)</td><td>Fisher Scientific</td><td>BP328-500</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>Fisher Scientific</td><td>S318-500</td><td></td></tr><tr><td>Soybean Trypsin Inhibitor &lt;sup&gt;8&lt;/sup&gt;</td><td>Gibco</td><td>17075029</td><td></td></tr><tr><td>Spatula</td><td>Fisherbrand</td><td>21-401-10</td><td></td></tr><tr><td>Steriflip 50 mL, 0.22 &amp;mu;m filters&amp;nbsp;</td><td>Millipore</td><td>SCGP00525</td><td></td></tr><tr><td>TGF-&amp;alpha;</td><td>R&amp;amp;D Systems</td><td>239-A-100</td><td></td></tr><tr><td>Type I Rat Tail Collagen</td><td>Corning</td><td>354236</td><td></td></tr><tr><td>Waymouth MB 752/1 Medium (powder)</td><td>Sigma</td><td>W1625-10X1L</td><td></td></tr><tr><td>Weigh boat, hexagonal, medium&amp;nbsp;</td><td>Fisherbrand</td><td>02-202-101</td><td></td></tr></tbody></table>

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.

Completion of the protocol herein occurs within one day and upon stimulation, ADM is seen in 3-5 days. Figure 1 depicts the sequence of the method, whereby steps 1 through 4 are completed on the first day. This includes preparation, acinar isolation, viral infection and embedment in collagen or basement membrane matrix. While the basement membrane matrix induces ADM, acinar cells within collagen I require a stimulus, such as TGF- &#945;, to undergo differenti...

Watch this Scientific Journal Video about Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture at JoVE.com

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

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 ...

mimicking-manipulating-pancreatic-acinar-to-ductal-metaplasia-3

Research

JoVE Journal

Cancer Research

8.6K Views.  Mayo Clinic Florida. 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.

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

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Developmental Biology

Phospholipid Mediator Induced Transformation in Three-Dimensional Cultures

Several models have been developed to study cancer, such as rodent models and established cell lines. Valuable insights into carcinogenesis have been provided by studies using these models. Cell lines have provided an understanding of the deregulation of molecular signaling associated with breast tumorigenesis, while rodent models are widely used to study cellular and molecular characteristics of breast cancer in vivo. The establishment of 3D cultures of breast epithelial and cancerous cells aids in bridging the gap between in vivo and in vitro models by mimicking the in vivo conditions in vitro. This model can be used to understand the deregulation of complex molecular signaling events and the cellular characteristics during breast carcinogenesis. Here, a 3D culture system is modified to study a phospholipid mediator-induced (Platelet Activating Factor, PAF) transformation. Immunomodulators and other secreted molecules play a major role in tumor initiation and progression in the breast. In the present study, 3D acinar cultures of breast epithelial cells are exposed to PAF exhibited transformation characteristics such as loss of polarity and altered cellular characteristics. This 3D culture system will assist in shedding light on genetic and/or epigenetic perturbations induced by various small molecule entities in the tumor microenvironment. Additionally, this system will also provide a platform for the identification of novel as well as known genes that may be involved in the process of transformation.

The present protocol describes the setting up of 3D 'on top' cultures of a non-transformed breast epithelial cell line, MCF10A, that has been modified to study Platelet Activating Factor (PAF) induced transformation. Immuno-fluorescence has been used to assess the transformation and is discussed in detail.

Several models have been developed to study cancer, such as rodent models and established cell lines. Valuable insights into carcinogenesis have been ...

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

Medicine

Syngeneic Mouse Orthotopic Allografts to Model Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is a very complex disease characterized by a heterogeneous tumor microenvironment made up of a diverse stroma, immune cells, vessels, nerves, and extracellular matrix components. Over the years, different mouse models of PDAC have been developed to address the challenges posed by its progression, metastatic potential, and phenotypic heterogeneity. Immunocompetent mouse orthotopic allografts of PDAC have shown good promise owing to their fast and reproducible tumor progression in comparison to genetically engineered mouse models. Moreover, combined with their ability to mimic the biological features observed in autochthonous PDAC, cell line-based orthotopic allograft mouse models enable large-scale in vivo experiments. Thus, these models are widely used in preclinical studies for rapid genotype-phenotype and drug-response analyses. The aim of this protocol is to provide a reproducible and robust approach to successfully inject primary mouse PDAC cell cultures into the pancreas of syngeneic recipient mice. In addition to the technical details, important information is given that must be considered before performing these experiments.

Syngeneic mouse orthotopic allografts of pancreatic ductal adenocarcinoma (PDAC) recapitulate the biology, phenotypes, and therapeutic responses of disease subtypes. Owing to their fast, reproducible tumor progression, they are widely used in preclinical studies. Here, we show common practices to generate these models, injecting syngeneic murine PDAC cultures into the pancreas.

Pancreatic ductal adenocarcinoma (PDAC) is a very complex disease characterized by a heterogeneous tumor microenvironment made up of a diverse stroma, ...

3D-PDAC Model Capturing Desmoplasia for Therapeutic Evaluation

Growing Desmoplastic Three-Dimensional Pancreatic Cancer Spheroids from Co-Culture

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with a 5-year survival rate of &#60;12%. The biggest barrier to therapy is the dense desmoplastic extracellular matrix (ECM) that surrounds the tumor and reduces vascularization, generally termed desmoplasia. A variety of drug combinations and formulations have been tested to treat the cancer, and although many of them show success pre-clinically, they fail clinically. It, therefore, becomes important to have a clinically relevant model available that can predict the response of the tumor to therapy. This model has been previously validated against resected clinical tumors. Here a simple protocol to grow desmoplastic three-dimensional (3D)-coculture spheroids is described that can naturally generating a robust ECM and do not require any external matrix sources or scaffold to support their growth.
Briefly human pancreatic stellate cells (HPaSteC) and PANC-1 cells are used to prepare a suspension containing the cells in a 1:2 ratio, respectively. The cells are plated in a poly-HEMA coated, 96-well low attachment U-well plate. The plate is centrifuged to allow the cells to form an initial pellet. The plate is stored in the incubator at 37 &#176;C with 5% CO2, and media is replaced every 3 days. Plates can be imaged at designated intervals to measure spheroid volume. Following 14 days of culture, mature desmoplastic spheroids are formed (i.e. average volume of 0.048 + 0.012 mm3&#160;(451 &#181;m x 462.84 &#181;m)) and can be utilized for experimental therapy assessment. Mature ECM components include collagen-I, hyaluronic acid, fibronectin, and laminin.

Author Spotlight: Validating Cancer Therapy Responses with Desmoplastic Spheroid Models

Pancreatic cancer remains one of the toughest cancers to treat. Therefore, it is critical that pre-clinical models evaluating treatment efficacy are reproducible and clinically relevant. This protocol describes a simple co-culture procedure to generate reproducible, clinically relevant desmoplastic spheroids.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with a 5-year survival rate of &#60;12%. The biggest barrier to therapy is the ...

A System for ex vivo Culturing of Embryonic Pancreas

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

A System for ex vivo Culturing of Embryonic Pancreas

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

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

Biology

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.

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 ...

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 ...

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 ...

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.

Indukcja metaplazji acinar-przewodowa: metoda badania różnicowania komórek groniastych do komórek przewodowych w hodowli 3D ex vivo

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

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

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Phospholipid Mediator Induced Transformation in Three-Dimensional Cultures

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Syngeneic Mouse Orthotopic Allografts to Model Pancreatic Cancer

Growing Desmoplastic Three-Dimensional Pancreatic Cancer Spheroids from Co-Culture

A System for ex vivo Culturing of Embryonic Pancreas

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

De Novo Generation of Somatic Stem Cells by YAP/TAZ

Indukcja metaplazji acinar-przewodowa: metoda badania różnicowania komórek groniastych do komórek przewodowych w hodowli 3D ex vivo