Name: whole cell electrophysiology of primary cultured murine enterochromaffin cells
Uploaded: 2018-09-26T13:30:00
Description: Watch this Scientific Journal Video about Whole Cell Electrophysiology of Primary Cultured Murine Enterochromaffin Cells at JoVE.com

The authors thank Mrs. Lyndsay Busby for administrative assistance and Mr. Robert Highet from Mayo Clinic Division of Engineering for design and 3D printing of the culture dish insert. This work was supported by NIH K08 to AB (DK106456), Pilot and Feasibility Grant to AB from Mayo Clinic Center for Cell Signaling in Gastroenterology (NIH P30DK084567) and 2015 American Gastroenterological Association Research Scholar Award (AGA RSA) to AB and NIH R01 to GF (DK52766).

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Mayo Clinic. The primary cell culture portion of this protocol is based on previously published methods that can be referenced for further details10. All experimental procedures must be approved by the (IACUC), and all experiments must be performed in accordance with relevant guidelines and regulations.
1. Culture Preparation
<ol>
	<li>Media.
	<...

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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+channel+NaV1.3+is+important+for+enterochromaffin+cell+excitability+and+serotonin+release.">Sodium channel NaV1.3 is important for enterochromaffin cell excitability and serotonin release.</a> Scientific Reports. 7 (15650), (2017).</li><li>Bellono, N. W., 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=Enterochromaffin+cells+are+gut+chemosensors+that+couple+to+sensory+neural+pathways.">Enterochromaffin cells are gut chemosensors that couple to sensory neural pathways.</a> Cell. 170 (1), 185-198 (2017).</li><li>Alcaino, C., Knutson, K., Gottlieb, P. A., Farrugia, G., Beyder, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanosensitive+ion+channel+Piezo2+is+inhibited+by+D-GsMTx4.">Mechanosensitive ion channel Piezo2 is inhibited by D-GsMTx4.</a> Channels. 11 (3), 245-253 (2017).</li><li>Kim, M., Javed, N. H., Yu, J. G., Christofi, F., Cooke, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+stimulation+activates+Galphaq+signaling+pathways+and+5-hydroxytryptamine+release+from+human+carcinoid+BON+cells.">Mechanical stimulation activates Galphaq signaling pathways and 5-hydroxytryptamine release from human carcinoid BON cells.</a> Journal of Clinical Investigation. 108 (7), 1051-1059 (2001).</li><li>Modlin, I. M., Kidd, M., Pfragner, R., Eick, G. N., Champaneria, M. 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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=Distinct+cellular+origins+for+serotonin-expressing+and+enterochromaffin-like+cells+in+the+gastric+corpus.">Distinct cellular origins for serotonin-expressing and enterochromaffin-like cells in the gastric corpus.</a> Gastroenterology. 146 (3), 754-764 (2014).</li><li>Psichas, A., Tolhurst, G., Brighton, C. A., Gribble, F. M., Reimann, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mixed+primary+cultures+of+murine+small+intestine+intended+for+the+study+of+gut+hormone+secretion+and+live+cell+imaging+of+enteroendocrine+cells.">Mixed primary cultures of murine small intestine intended for the study of gut hormone secretion and live cell imaging of enteroendocrine cells.</a> Journal of Visualized Experiments. (122), 55687 (2017).</li><li>Raghupathi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+unique+release+kinetics+of+serotonin+from+guinea-pig+and+human+enterochromaffin+cells.">Identification of unique release kinetics of serotonin from guinea-pig and human enterochromaffin cells.</a> The Journal of Physiology. 591, 5959-5975 (2013).</li><li>Nozawa, K., 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=TRPA1+regulates+gastrointestinal+motility+through+serotonin+release+from+enterochromaffin+cells.">TRPA1 regulates gastrointestinal motility through serotonin release from enterochromaffin cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (9), 3408-3413 (2009).</li><li>Reimann, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+functional+role+of+voltage+gated+cation+conductances+in+the+glucagon-like+peptide-1+secreting+GLUTag+cell+line.">Characterization and functional role of voltage gated cation conductances in the glucagon-like peptide-1 secreting GLUTag cell line.</a> The Journal of Physiology. 563, 161-175 (2005).</li></ol>

Fully understanding EC cell function requires a high-quality primary culture method to generate cells for whole cell electrophysiology. Primary cultures of GI epithelium had traditionally been difficult due to low survival. Alleviating this confounding factor, the present method yields cultures of singular EC cells from either small or large bowel that are capable of surviving for several days.
We found that the following were critical for improving the quality of cultures to be suitable for e...

The gastrointestinal (GI) epithelium is a diverse community consisting of several cell types. Enteroendocrine cells comprise roughly 1% of all epithelial cells, and enterochromaffin (EC) cells are the largest enteroendocrine cell population1. Recent studies show that enteroendocrine2 and EC3,4 cells are electrically excitable. We are interested in understanding primary EC cell electrophysiology. Thus, the purpose of this study was to establish primary EC cell cultures optimized for whole cell electrophysiology.
Existing EC cell lines that produce and secrete 5-HT (e.g.,&#160;QGP-15, BON6, KRJ-17) and have been used to examine electrophysiology5,8 are typically generated from immortalized neoplastic tissues. While the information acquired from these cell lines is valuable5,8, studies of primary cell electrophysiology are necessary to properly understand EC cell physiology. The electrophysiology of primary EC cells requires the isolation and culture of single epithelial cells, which has been limited by low viability of epithelial cultures.
The culture method presented in this study relies on transgenic mice with fluorescently labeled EC cells, like Tph1-CFP9, used in this study. The method optimizes mixed primary epithelial cultures developed previously2,10 for single cell electrophysiology3. Previous methods used combinations of Trypsin/EDTA plus Collagenase A or EDTA and DTT for enzymatic digestion and used density gradients to specifically isolate and culture guinea-pig11 and rat EC cells12. More recently, intestinal organoids were generated and mechanically disrupted for electrophysiological recordings4. Cultures using these methods are well suited for serotonin release experiments, RT-qPCR analysis and, while they could be used for electrophysiology, are reliant on the success of time consuming organoid generation, density gradients to identify EC cells, and the cellular disruptive effects of Trypsin and EDTA. By contrast, the protocol described here improves enzymatic and mechanical treatments, optimizes culturing conditions, and streamlines procedures to produce single and healthy EC cells suitable for the high cellular standards needed for electrophysiology.
This method will be useful for scientists who want to work on primary EC cells in mixed cultures rather than immortalized cultures and want to investigate the electrophysiology of single cells. However, it may prove less appropriate for studying cell populations that need sorting or long-term cultures that require survival past 72 h.

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			<td height="20" style="height:20px;width:276px;">High Glucose DMEM</td>
			<td style="width:179px;">Sigma</td>
			<td style="width:175px;">D6546</td>
			<td style="width:564px;">Store at 4&#730; C for no longer than 1 month</td>
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			<td height="20" style="height:20px;width:276px;">Fetal Bovine Serum (FBS), Heat Inactivated</td>
			<td style="width:179px;">Sigma</td>
			<td style="width:175px;">F4135</td>
			<td>Freeze in 25 mL aliquots&#160; and store long term at -20&#730; C</td>
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			<td height="20" style="height:20px;width:276px;">L-Glutamine</td>
			<td style="width:179px;">Life Technologies</td>
			<td style="width:175px;">25030-081</td>
			<td>Freeze in 5 mL aliquots and store long term at -20&#730; C</td>
		</tr>
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			<td height="20" style="height:20px;width:276px;">Penicillin/ Streptomycin</td>
			<td style="width:179px;">Sigma</td>
			<td style="width:175px;">P0781</td>
			<td>Freeze in 5 mL aliquots and store long term at -20&#730; C</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:179px;">Sigma</td>
			<td style="width:175px;">A2153</td>
			<td>Store at 4&#730; C for long term storage</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Collagenase XI</td>
			<td style="width:179px;">Sigma</td>
			<td style="width:175px;">C9407</td>
			<td>Store at -20&#730; C in aliquots of 100mg, avoid freeze thaw and avoid lot to lot variation</td>
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			<td height="20" style="height:20px;width:276px;">Phosphate Buffered Saline (PBS), w/ Ca and Mg</td>
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			<td height="20" style="height:20px;width:276px;">Y-27632 (Rock Inhibitor)</td>
			<td style="width:179px;">StemCell</td>
			<td style="width:175px;">72304</td>
			<td>Resuspend at 5mM and store in small aliquots at -80&#730; C</td>
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			<td height="20" style="height:20px;width:276px;">100x15mm culture dish</td>
			<td style="width:179px;">Corning (Falcon)</td>
			<td>351029</td>
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			<td height="20" style="height:20px;width:276px;">Transfer Pipette</td>
			<td style="width:179px;">Fisherbrand</td>
			<td style="width:175px;">13-711-7M</td>
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			<td height="20" style="height:20px;width:276px;">10 mL serological pipette</td>
			<td style="width:179px;">Corning (Falcon)&#160;</td>
			<td style="width:175px;">357551</td>
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			<td height="20" style="height:20px;width:276px;">50 mL Conical</td>
			<td style="width:179px;">Corning (Falcon)&#160;</td>
			<td style="width:175px;">352098</td>
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			<td height="20" style="height:20px;width:276px;">15 mL Conical</td>
			<td style="width:179px;">Corning (Falcon)&#160;</td>
			<td style="width:175px;">352097</td>
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			<td height="20" style="height:20px;width:276px;">1000 mL Barrier Pipet Tips</td>
			<td style="width:179px;">Thermo Scientific (Art)</td>
			<td style="width:175px;">2079E</td>
			<td>Keep sterile</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Matrigel, Growth Factor Reduced</td>
			<td style="width:179px;">Corning</td>
			<td style="width:175px;">354230</td>
			<td>Store at 150uL aliquots at -20&#730; C. Thaw on ice and keep cold until ready for plating</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:276px;">MatTek Glass Bottom Dishes</td>
			<td style="width:179px;">MatTek Corporation</td>
			<td style="width:175px;">P35G-1.5-14-C</td>
			<td>Keep sterile</td>
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		<tr height="35">
			<td height="35" style="height:35px;width:276px;">Micro-dissection Forceps - Very Fine, Extra-Long Points</td>
			<td style="width:179px;">Fine Science Tools</td>
			<td>SB12630M</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Surgical Scissors-Sharp</td>
			<td style="width:179px;">Nasco</td>
			<td>14002-14</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Micro-Dissection Scissors</td>
			<td style="width:179px;">Nasco</td>
			<td style="width:175px;">SB46568M</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Monoject Hypo Needle, 21G x 1&#34; A, 100/BX</td>
			<td style="width:179px;">Covidien</td>
			<td style="width:175px;">8881250172</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:276px;">&#160;Tg(Tph1-CFP)1Able/J Mice</td>
			<td style="width:179px;">Jackson</td>
			<td>Stock # 028366</td>
			<td>Use male or female mice between the ages of 4-7 weeks</td>
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		<tr height="35">
			<td height="35" style="height:35px;width:276px;">Microfil nonmetallic syringe needles (34 gauge, 67 mm)</td>
			<td style="width:179px;">World Precision Instruments</td>
			<td>MF34G-5</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Parafilm &#34;M&#34; Laboratory Film</td>
			<td style="width:179px;">Pechiney Plastic Packaging</td>
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			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:276px;">R6101</td>
			<td style="width:179px;">Dow Corning</td>
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			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:276px;">6-mL syringe with regular luer tip</td>
			<td style="width:179px;">Monoject</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">3-mL syringe with regular luer tip</td>
			<td style="width:179px;">Monoject</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Electrophysiology Equipment</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Amplifier</td>
			<td style="width:179px;">Molecular Devices</td>
			<td>Axopatch 200B</td>
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			<td height="20" style="height:20px;width:276px;">Data acquisition system (daq)</td>
			<td style="width:179px;">Molecular Devices</td>
			<td>Digidata 1440A</td>
			<td></td>
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			<td height="20" style="height:20px;width:276px;">Signal conditioner</td>
			<td style="width:179px;">Molecular Devices</td>
			<td>CyberAmp 320</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Software</td>
			<td style="width:179px;">Molecular Devices</td>
			<td>pClamp 10.6</td>
			<td></td>
		</tr>
	</tbody>
</table>

whole cell electrophysiology of primary cultured murine enterochromaffin cells

Enterochromaffin (EC) cells in the gastrointestinal (GI) epithelium constitute the largest subpopulation of enteroendocrine cells. As specialized sensory cells, EC cells sense luminal stimuli and convert them into serotonin (5-hyroxytryptamine, 5-HT) release events. However, the electrophysiology of these cells is poorly understood because they are difficult to culture and to identify. The method presented in this paper outlines primary EC cell cultures optimized for single cell electrophysiology. This protocol utilizes a transgenic cyan fluorescent protein (CFP) reporter to identify mouse EC cells in mixed primary cultures, advancing the approach to obtaining high-quality recordings of whole cell electrophysiology in voltage- and current-clamp modes.

Cultures: 
Primary murine epithelial cells made from a transgenic Tph1-CFP model attach to dishes after 4 hours and are ready for physiological experiments between 24 to 72 h. When culture conditions had not been optimized, the epithelial cell culture consisted of large clumps, floating cellular debris, damaged membranes, and weak CFP signal in the EC cells (Figure 1A). Cultures that have been optimized for electrophysio...

Watch this Scientific Journal Video about Whole Cell Electrophysiology of Primary Cultured Murine Enterochromaffin Cells at JoVE.com

Whole Cell Electrophysiology of Primary Cultured Murine Enterochromaffin Cells

Enterochromaffin (EC) cells comprise a small subset of gastrointestinal epithelial cells. EC cells are electrically excitable and release serotonin, yet difficulties in culturing and identifying EC cells have limited physiological studies. The method presented here establishes a primary culture model amenable to examination of single EC cells by electrophysiology.

Enterochromaffin (EC) cells in the gastrointestinal (GI) epithelium constitute the largest subpopulation of enteroendocrine cells. As specialized sensory ...

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