Name: mouse- and human-derived primary gastric epithelial monolayer culture for the study of regeneration
Uploaded: 2018-05-07T13:30:00
Description: Watch this Scientific Journal Video about Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration at JoVE.com

This work was supported by NIH (NIDDK) 5 R01 DK083402-07 grant (YZ).

To avoid contamination, perform protocol in its entirety within a sterile tissue culture hood. Human fundic tissue was collected from patients undergoing sleeve gastrectomy according to the approved University of Cincinnati IRB protocol (IRB protocol number: 2015 - 4869). 
All mouse studies were approved by the University of Cincinnati Institutional Animal Care and Use Committee (IACUC) that maintains an American Association of Assessment and Accreditation of Laboratory Animal Care (AAALAC) facility.
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<ol>
	<li>Nagy, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicobacter+pylori+regulates+cellular+migration+and+apoptosis+by+activation+of+phosphatidylinositol+3-kinase+signaling.">Helicobacter pylori regulates cellular migration and apoptosis by activation of phosphatidylinositol 3-kinase signaling.</a> J Infect Dis. 199 (5), 641-651 (2009).</li><li>Mnich, E., 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=Impact+of+Helicobacter+pylori+on+the+healing+process+of+the+gastric+barrier.">Impact of Helicobacter pylori on the healing process of the gastric barrier.</a> World J Gastroenterol. 22 (33), 7536-7558 (2016).</li><li>Zhang, 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=Bm-TFF2,+a+toad+trefoil+factor,+promotes+cell+migration,+survival+and+wound+healing.">Bm-TFF2, a toad trefoil factor, promotes cell migration, survival and wound healing.</a> Biochem Biophys Res Commun. 398 (3), 559-564 (2010).</li><li>Crabtree, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+cytokines+in+pathogenesis+of+Helicobacter+pylori-induced+mucosal+damage.">Role of cytokines in pathogenesis of Helicobacter pylori-induced mucosal damage.</a> Dig Dis Sci. 43 (Supplement), 46S-53S (1998).</li><li>Riahi, R., Yang, Y., Zhang, D. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+wound-healing+assays+for+probing+collective+cell+migration.">Advances in wound-healing assays for probing collective cell migration.</a> J Lab Autom. 17 (1), 59-65 (2012).</li><li>Zhang, M., Li, H., Ma, H., Qin, 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+simple+microfluidic+strategy+for+cell+migration+assay+in+an+in+vitro+wound-healing+model.">A simple microfluidic strategy for cell migration assay in an in vitro wound-healing model.</a> Wound Repair Regen. 21 (6), 897-903 (2013).</li><li>Schlaermann, P., 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=A+novel+human+gastric+primary+cell+culture+system+for+modelling+Helicobacter+pylori+infection+in+vitro.">A novel human gastric primary cell culture system for modelling Helicobacter pylori infection in vitro.</a> Gut. 65 (2), 202-213 (2016).</li><li>Bertaux-Skeirik, 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=CD44+plays+a+functional+role+in+Helicobacter+pylori-induced+epithelial+cell+proliferation.">CD44 plays a functional role in Helicobacter pylori-induced epithelial cell proliferation.</a> PLoS Pathog. 11 (2), e1004663 (2015).</li><li>Tarnawski, A. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histology+of+the+stomach+and+duodenum+in+Crohn's+disease.">Histology of the stomach and duodenum in Crohn's disease.</a> Am J Surg Pathol. 22 (4), 383-390 (1998).</li><li>Engevik, A. 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=The+Development+of+Spasmolytic+Polypeptide/TFF2-Expressing+Metaplasia+(SPEM)+During+Gastric+Repair+Is+Absent+in+the+Aged+Stomach.">The Development of Spasmolytic Polypeptide/TFF2-Expressing Metaplasia (SPEM) During Gastric Repair Is Absent in the Aged Stomach.</a> CMGH. , (2016).</li><li>Bertaux-Skeirik, 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=CD44+variant+isoform+9+emerges+in+response+to+injury+and+contributes+to+the+regeneration+of+the+gastric+epithelium.">CD44 variant isoform 9 emerges in response to injury and contributes to the regeneration of the gastric epithelium.</a> Journal of Pathology. 242 (4), 463-475 (2017).</li><li>Nam, K. 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The current protocol details the establishment of human- and mouse-derived gastric epithelial monolayers that can be used for scratch-wound assays. The protocol depends on the concept of resident stem cell isolation from primary human (or mouse) tissues and is a modified protocol first published by Schlaermann et al7. In particular, we have optimized the protocol here to establish a confluent and polarized gastric epithelial monolayer which expresses the major cell lineages which can be f...

The use of scratch-wound assays is a popular method for studying repair and regeneration1,2,3,4,5,6. The proposed methodology may be used to specifically study gastric regeneration and Helicobacter pylori colonization. In the past, gastric cancer cell lines have been used as a means to study Helicobacter pylori (H. pylori) infection1,2 and up until recently gastric cancer cell lines such as AGS cells were favored1,2,3,4. One limitation of the gastric cancer cell cultures is their failure to recapitulate the cellular diversity of the gastric epithelium. To try to address this limitation, demonstrated here is the establishment and transfer of primary human- and mouse-derived 3-dimensional fundic gastric organoids (FGOs) to a gastric epithelial monolayer for wound healing assays based on a modified method first described by Schlaermann et al.7 We demonstrate that gastric epithelial monolayers derived from sheared 3D gastric FGOs or FGO-derived single cells retain a polarized cellular composition that closely represents that of the gastric epithelium in vivo. Given that gastric cancer cell lines do not demonstrate the cell composition this technique displays, the current protocol has an advantage over alternative scratch-wound assay methods. This methodology has been optimized whereby monolayers can be established from whole organoids or from a single cell suspension from dissociated organoids and plated using a basement membrane matrix or collagen-coating. The overall goal of this protocol is to establish an organoid derived gastric epithelial monolayer cultures for a novel wound healing assay.

<table>
	<tbody>
		<tr>
			<td>Advanced Dulbecco&#39;s modified Eagle/F12 medium (basal media)</td>
			<td>Life Technologies</td>
			<td>12634-010</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX (L-glutamine)</td>
			<td>Life Technologies</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Thermo Scientific</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B/Gentamicin</td>
			<td>Thermo Scientific</td>
			<td>R01510</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Thermo Fisher</td>
			<td>RO1510</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES Buffer</td>
			<td>Sigma Aldrich</td>
			<td>H0887</td>
			<td></td>
		</tr>
		<tr>
			<td>n-Acetylcystine</td>
			<td>Sigma Aldrich</td>
			<td>A9165</td>
			<td></td>
		</tr>
		<tr>
			<td>N2</td>
			<td>Life Technologies</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Life Technologies</td>
			<td>12587-010</td>
			<td></td>
		</tr>
		<tr>
			<td>BMP Inhibitor (Noggin)</td>
			<td>Pepro Tech</td>
			<td>250-38</td>
			<td></td>
		</tr>
		<tr>
			<td>Gastrin</td>
			<td>Tocris</td>
			<td>3006</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Pepro Tech</td>
			<td>315-09</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF10</td>
			<td>Pepro Tech</td>
			<td>100-26</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma Aldrich</td>
			<td>N0636</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632 ROCK Inhibitor</td>
			<td>Sigma Aldrich</td>
			<td>Y0503</td>
			<td></td>
		</tr>
		<tr>
			<td>TGF-&#946; Inhibitor</td>
			<td>Tocris</td>
			<td>2939</td>
			<td></td>
		</tr>
		<tr>
			<td>Ca2+/Mg2+ -free DPBS</td>
			<td>Fisher</td>
			<td>21-031CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle Medium (DMEM)</td>
			<td>Life Technologies</td>
			<td>12634-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>FBS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>OPTIMEM</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.22&#181;M filter</td>
			<td>Fisher</td>
			<td>099-720-004</td>
			<td></td>
		</tr>
		<tr>
			<td>37 &#176;C Water Bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type 1</td>
			<td>Worthington&#160;</td>
			<td>LS004214</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma Aldrich</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes (tissue paper)</td>
			<td>Fischer Scientific</td>
			<td>06-666C</td>
			<td></td>
		</tr>
		<tr>
			<td>125 mL round bottom flask</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 in (25 mm) stir bar</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rubber Septa</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Gauze</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stir Plate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ring Stand</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ring Stand Clamps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile petri dish</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>18 G needles of 1.5 in length</td>
			<td>Thermo Fisher</td>
			<td>305196</td>
			<td></td>
		</tr>
		<tr>
			<td>20 G spinal needles of 3.5 in length</td>
			<td>Thermo Fisher</td>
			<td>405182</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well cell culture treated plate</td>
			<td>Midwest Scientific</td>
			<td>92012</td>
			<td></td>
		</tr>
		<tr>
			<td>Growth Factor Reduced, Phenol Red-free MatrigelTM (Basement membrane matrix)</td>
			<td>Fisher</td>
			<td>CB-40230C</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Razor Blades</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wide-tip tweezers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Hemostatic Forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;L wide pipette tips</td>
			<td>Thermo Fisher</td>
			<td>02-707-134</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL cell culture test tubes</td>
			<td>Fisher</td>
			<td>14-956-3C</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen Tank with rubber hosing</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 g D-Sorbitol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher</td>
			<td>SS-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Tray</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rocking Table</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rat Tail Collagen Type 1</td>
			<td>Life Technologies</td>
			<td>A10483-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture grade glacial acetic acid</td>
			<td>Fisher</td>
			<td>A38-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture grade water</td>
			<td>Corning</td>
			<td>25-055-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>2-well chamber slide</td>
			<td>Thermo Fisher</td>
			<td>155380</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well Transwell (polyester membrane inserts)</td>
			<td>Corning</td>
			<td>3460</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>Corning</td>
			<td>353086</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase (cell detachment solution)</td>
			<td>Stemcell Technologies</td>
			<td>7920</td>
			<td></td>
		</tr>
		<tr>
			<td>263/8 G syringe</td>
			<td>Thermo Fisher</td>
			<td>309625</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L Cells</td>
			<td>L cells, a Wnt3a producing cell line, were received as a gift from Dr. Hans Clevers (Hubrecht Institute for Developmental Biology and Stem Cell Research, Netherlands).&#160;&#160;</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK-293T Rspondin secreting cells</td>
			<td>A modified HEK-293T R-spondin secreting cell line was obtained from Dr. Jeff Whitsett (Section of Neonatology, Perinatal and Pulmonary Biology, Cincinnati Children&#39;s Hospital Medical Centre and The University of Cincinnati College of Medicine, Cincinnati, USA&#160;).&#160;&#160;</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>HK-ATPase Primary Antibody</td>
			<td>Invitrogen</td>
			<td>SC-374094</td>
			<td></td>
		</tr>
		<tr>
			<td>E-Cadherinn</td>
			<td>SantaCruz</td>
			<td>sc-59778</td>
			<td></td>
		</tr>
		<tr>
			<td>UEA1</td>
			<td>Sigma Aldrich</td>
			<td>L9006</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Thermo Fisher</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexafluor 488 secondary antibody (Donkey anti-mouse 488 secondary antibody)</td>
			<td>ThermoFisher Scientific</td>
			<td>R37114</td>
			<td></td>
		</tr>
	</tbody>
</table>

mouse- and human-derived primary gastric epithelial monolayer culture for the study of regeneration

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and migration. One critical limitation of such assays, however, is their homogenous assortment of cellular types. Repair is a complex process which demands the interaction of several cell types. Therefore, to study a culture devoid of cellular subtypes, is a concern that must be overcome if we are to understand the repair process. The gastric organoid model may alleviate this issue whereby the heterogeneous collection of cell types closely reflects that of the gastric epithelium or other native tissues in vivo. Demonstrated here is a novel, in vitro scratch-wound assay derived from human or mouse 3-dimensional organoids which can then be transferred to a gastric epithelial monolayer as either intact organoids or as a single cell suspension of dissociated organoids. The goal of the protocol is to establish organoid-derived gastric epithelial monolayers that can be used in a novel scratch-wound assay system to study gastric regeneration.

Organoids were derived from either the corpus/body of the human or fundus of the mouse stomach tissues (Figure 1A). After either collagenase or EDTA digestion of human- or mouse-derived stomach tissue respectively, glands are embedded into basement membrane matrix and cultured for 6 - 7 days (Figure 1A). Figure 1B demonstrates the formation of human-derived gastric organoids (huFGOs) that have then b...

Watch this Scientific Journal Video about Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration at JoVE.com

Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration

Here we describe a protocol for establishing and culturing human- and mouse-derived 3-dimensional (3D) gastric organoids, and the method for the transfer of 3D organoids to a 2-dimensional monolayer. The use of the gastric epithelial monolayer as a novel scratch-wound assay for regeneration studies is also described.

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and ...

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