Name: mimicking and manipulating pancreatic acinar-to-ductal metaplasia in 3-dimensional cell culture
Uploaded: 2019-02-11T14:40:00
Description: Watch this Scientific Journal Video about Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture at JoVE.com

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...

<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 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 border="0" cellpadding="0" cellspacing="0" style="width:2181px;" width="2179">
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	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:357px;">37 &#176;C shaking incubator</td>
			<td style="width:167px;">Thermo Scientific</td>
			<td style="width:171px;">SHKE4000-7</td>
			<td style="width:1487px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:357px;">5% CO2, 37 &#176;C Incubator</td>
			<td style="width:167px;">NUAIRE</td>
			<td style="width:171px;">NU-5500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">50 ml tubes</td>
			<td style="width:167px;">Falcon</td>
			<td style="width:171px;">352070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Absorbent pad, 20&#34; x 24&#34;</td>
			<td style="width:167px;">Fisherbrand</td>
			<td style="width:171px;">1420662</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Adenovirus, Ad-GFP</td>
			<td style="width:167px;">Vector Biolabs&#160;</td>
			<td style="width:171px;">1060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Aluminum foil</td>
			<td style="width:167px;">Fisherbrand</td>
			<td style="width:171px;">01-213-101</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">BD Precision Glide Needle 21G x 1/2</td>
			<td style="width:167px;">Fisher Scientific&#160;</td>
			<td style="width:171px;">305167</td>
			<td>Use as pins for dissection&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Beaker, 600 mL</td>
			<td style="width:167px;">Fisherbrand</td>
			<td style="width:171px;">FB-101-600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Bleach, 8.25%</td>
			<td style="width:167px;">Clorox</td>
			<td style="width:171px;">31009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Cell Recovery Solution</td>
			<td style="width:167px;">Corning</td>
			<td style="width:171px;">354253</td>
			<td>Referred to as &#39;basement membrane matrix recovery solution&#39; in the manuscript.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Centrifuge&#160;</td>
			<td style="width:167px;">Beckman Coulter</td>
			<td style="width:171px;">Allegra X-15R Centrifuge</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Collagenase Type I, Clostridium histolyticum</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:171px;">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 &#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Dexamethasone</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:171px;">D1756</td>
			<td>Create a 4 mg/ml solution by dissolving powder in methanol, aliquoting and storing at -20 &#176;C.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Ethanol, 200 proof</td>
			<td style="width:167px;">Decon Laboratories&#160;</td>
			<td style="width:171px;">2701</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Fetal Bovine Serum&#160;</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:171px;">F0926-100mL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Forceps&#160;</td>
			<td style="width:167px;">Fine Science Tools</td>
			<td style="width:171px;">11002-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Forceps&#160;</td>
			<td style="width:167px;">Fine Science Tools</td>
			<td style="width:171px;">91127-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Glass slide, 8-well</td>
			<td style="width:167px;">Lab-Tek</td>
			<td style="width:171px;">177402</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:357px;">Hank&#39;s Balanced Salt Solution (HBSS), No calcium, No magnesium, No phenol red</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:171px;">SH3048801</td>
			<td>&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Ice bucket, rectangular</td>
			<td style="width:167px;">Fisher Scientific&#160;</td>
			<td style="width:171px;">07-210-103</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Instant Sealing Sterilization Pouch</td>
			<td style="width:167px;">Fisherbrand</td>
			<td style="width:171px;">01-812-51</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:357px;">LAB GUARD specimen bags (for mouse after dissection)&#160;</td>
			<td style="width:167px;">Minigrip</td>
			<td style="width:171px;">SBL2X69S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Lentiviral Packaging Mix, Virapower</td>
			<td style="width:167px;">Invitrogen</td>
			<td style="width:171px;">44-2050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Matrigel</td>
			<td style="width:167px;">Corning</td>
			<td style="width:171px;">356234</td>
			<td>Referred to as &#39;basement membrane matrix&#39; in the manuscript.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Parafilm</td>
			<td style="width:167px;">Bemis</td>
			<td style="width:171px;">PM992</td>
			<td>Referred to as &#39;plastic paraffin film&#39; in the manuscript.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">PBS</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:171px;">SH30028.02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Penicillin-Streptomycin&#160;</td>
			<td style="width:167px;">ThermoFisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Pipet tips, 10 &#181;l</td>
			<td style="width:167px;">USA Scientific</td>
			<td style="width:171px;">1110-3700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Pipet tips, 1000 &#181;l&#160;</td>
			<td style="width:167px;">Olympus Plastics</td>
			<td style="width:171px;">24-165RL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Pipet tips, 200 &#181;l&#160;</td>
			<td style="width:167px;">USA Scientific</td>
			<td style="width:171px;">1111-1700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Pipet-Aid</td>
			<td style="width:167px;">Drummond</td>
			<td style="width:171px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">PIPETMAN Classic P10, 1-10 &#956;l</td>
			<td style="width:167px;">Gilson</td>
			<td style="width:171px;">F144802</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">PIPETMAN Classic P1000, 200-1000 &#956;l</td>
			<td style="width:167px;">Gilson</td>
			<td style="width:171px;">F123602</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">PIPETMAN Classic P20, 2-20 &#956;l</td>
			<td style="width:167px;">Gilson</td>
			<td style="width:171px;">F123600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">PIPETMAN Classic P200, 20-200 &#956;l</td>
			<td style="width:167px;">Gilson</td>
			<td style="width:171px;">F123601</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Pipettes, 10 ml&#160;</td>
			<td style="width:167px;">Falcon</td>
			<td style="width:171px;">357551</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Pipettes, 25 ml</td>
			<td style="width:167px;">Falcon</td>
			<td style="width:171px;">357525</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Pipettes, 5 ml&#160;</td>
			<td style="width:167px;">Falcon</td>
			<td style="width:171px;">357543</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Plate, 12-well</td>
			<td style="width:167px;">Corning Costar</td>
			<td style="width:171px;">3513</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Plate, 24-well plate</td>
			<td style="width:167px;">Corning Costar</td>
			<td style="width:171px;">3524</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Plate, 35 mm</td>
			<td style="width:167px;">Falcon</td>
			<td style="width:171px;">353001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Plate, 6-well</td>
			<td style="width:167px;">Falcon</td>
			<td style="width:171px;">353046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Polybrene</td>
			<td style="width:167px;">EMD Millipore</td>
			<td style="width:171px;">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 &#176;C.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Polypropylene Mesh, 105 &#181;m</td>
			<td style="width:167px;">Spectrum Labs</td>
			<td style="width:171px;">146436</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Polypropylene Mesh, 500 &#181;m&#160;</td>
			<td style="width:167px;">Spectrum Labs</td>
			<td style="width:171px;">146418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Scissors&#160;</td>
			<td style="width:167px;">Fine Science Tools</td>
			<td style="width:171px;">14568-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Scissors&#160;</td>
			<td style="width:167px;">Fine Science Tools</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Sodium Bicarbonate (Fine White Powder)</td>
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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 ...

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