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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Whole Cell Electrophysiology of Primary Cultured Murine Enterochromaffin Cells

Published: September 26th, 2018

DOI:

10.3791/58112

1Enteric Neuroscience Program, Division of Gastroenterology & Hepatology,Department of Physiology & Biomedical Engineering, Mayo Clinic, 2Division of Gastroenterology, Department of Medicine, University of Massachusetts Medical School
* These authors contributed equally

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 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.

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 t....

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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

  1. Media. <.......

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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.......

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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.......

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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).

....

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Name Company Catalog Number Comments
High Glucose DMEM Sigma D6546 Store at 4˚ C for no longer than 1 month
Fetal Bovine Serum (FBS), Heat Inactivated Sigma F4135 Freeze in 25 mL aliquots  and store long term at -20˚ C
L-Glutamine Life Technologies 25030-081 Freeze in 5 mL aliquots and store long term at -20˚ C
Penicillin/ Streptomycin Sigma P0781 Freeze in 5 mL aliquots and store long term at -20˚ C
Bovine Serum Albumin (BSA) Sigma A2153 Store at 4˚ C for long term storage
Collagenase XI Sigma C9407 Store at -20˚ C in aliquots of 100mg, avoid freeze thaw and avoid lot to lot variation
Phosphate Buffered Saline (PBS), w/ Ca and Mg Sigma D8662 Store long term at   4˚ C
Y-27632 (Rock Inhibitor) StemCell 72304 Resuspend at 5mM and store in small aliquots at -80˚ C
100x15mm culture dish Corning (Falcon) 351029
Transfer Pipette Fisherbrand 13-711-7M
10 mL serological pipette Corning (Falcon)  357551
50 mL Conical Corning (Falcon)  352098
15 mL Conical Corning (Falcon)  352097
1000 mL Barrier Pipet Tips Thermo Scientific (Art) 2079E Keep sterile
Matrigel, Growth Factor Reduced Corning 354230 Store at 150uL aliquots at -20˚ C. Thaw on ice and keep cold until ready for plating
MatTek Glass Bottom Dishes MatTek Corporation P35G-1.5-14-C Keep sterile
Micro-dissection Forceps - Very Fine, Extra-Long Points Fine Science Tools SB12630M
Surgical Scissors-Sharp Nasco 14002-14
Micro-Dissection Scissors Nasco SB46568M
Monoject Hypo Needle, 21G x 1" A, 100/BX Covidien 8881250172
 Tg(Tph1-CFP)1Able/J Mice Jackson Stock # 028366 Use male or female mice between the ages of 4-7 weeks
Microfil nonmetallic syringe needles (34 gauge, 67 mm) World Precision Instruments MF34G-5
Parafilm "M" Laboratory Film Pechiney Plastic Packaging
R6101 Dow Corning
6-mL syringe with regular luer tip Monoject
3-mL syringe with regular luer tip Monoject
Electrophysiology Equipment
Amplifier Molecular Devices Axopatch 200B
Data acquisition system (daq) Molecular Devices Digidata 1440A
Signal conditioner Molecular Devices CyberAmp 320
Software Molecular Devices pClamp 10.6

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