Working with Auditory HEI-OC1 Cells

Summary

House Ear Institute-Organ of Corti 1 (HEI-OC1) is one of the few mouse auditory cell lines currently available for research purposes. This protocol describes how to work with HEI-OC1 cells to investigate the cytotoxic effects of pharmacological drugs as well as functional properties of inner ear proteins.

Abstract

HEI-OC1 is one of the few mouse auditory cell lines available for research purposes. Originally proposed as an in vitro system for screening of ototoxic drugs, these cells have been used to investigate drug-activated apoptotic pathways, autophagy, senescence, mechanism of cell protection, inflammatory responses, cell differentiation, genetic and epigenetic effects of pharmacological drugs, effects of hypoxia, oxidative and endoplasmic reticulum stress, and expression of molecular channels and receptors. Among other several important markers of cochlear hair cells, HEI-OC1 cells endogenously express prestin, the paradigmatic motor protein of outer hair cells. Thus, they can be very useful to elucidate novel functional aspects of this important auditory protein. HEI-OC1 cells are very robust, and their culture usually does not present big complications. However, they require some special conditions such as avoiding the use of common anti-bacterial cocktails containing streptomycin or other antibiotics as well as incubation at 33 °C to stimulate cell proliferation and incubation at 39 °C to trigger cell differentiation. Here, we describe how to culture HEI-OC1 cells and how to use them in some typical assays, such as cell proliferation, viability, death, autophagy and senescence, as well as how to perform patch-clamp and non-linear capacitance measurements.

Introduction

House Ear Institute-Organ of Corti 1 (HEI-OC1) cells are derived from the auditory organ of a transgenic mouse ^1,2. Incubation of any cell from this transgenic mouse at 33 °C/10% CO₂ (permissive conditions) induces expression of an immortalizing gene that triggers de-differentiation and accelerated proliferation; moving the cells to 39 °C/5% CO₂ (non-permissive conditions) lead to decreased proliferation, differentiation and, at least in the case of HEI-OC1, cell death ^2,3.

HEI-OC1 cells were cloned and characterized in our laboratory over a decade ago, and initial studies indicated that....

Protocol

1. Cell Culture

Note: All cell culturing protocols must be performed using proper cell culture techniques (for reference see the first 3 Chapters of Cell Biology: A Laboratory Handbook, Volume I ¹⁰). HEI-OC1 cells do not require any additional coating or treatment of the cell culture dishes for proper adherence and growth. Very important: do not use glassware dishes for cell culture purposes; the phenotype and biological response of the cells to pharmacological drugs will change (G Kalinec & F Kalinec, unpublished); conventional plastic cell culture dishes are recommended (see Table of Materials/Equipment). Pay special attentio....

Results

In a couple of recent publications we reported a comprehensive set of studies aimed at evaluating the response of HEI-OC1 cells to several commonly used pharmacological drugs as well as investigating prestin function ^8,9. In these studies we made use of all the protocols described in the previous sections.

One of the results of these previous studies was that HEI-OC1 cells cultured at non-permissive conditions (39 °C/.......

Discussion

In this report we describe how to culture HEI-OC1 cells and use them to evaluate mechanisms of drug-induced cytotoxicity and to investigate functional properties of prestin, the molecular motor of cochlear OHCs. The technical procedures, however, are general enough to be easily adapted to different studies.

All the protocols described here require the correct use of well-established cell culture techniques ¹⁰. Just like with any other cell line, working with HEI-OC1 cells requires a.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
HEI-OC1 cells			ALL THE ASSAY KITS, EQUIPMENTS
Class II Biological Safety cabinet	The Baker Company	Sterilgard III	AND COMPANIES INDICATED IN THE
Refrigerated centrifuge	Eppendorf	5810R	PREVIOUS 2 COLUMNS ARE ONLY
Inverted microscope	Zeiss	Axiovert 25	EXAMPLES, AND ANY OTHER SIMILAR
Waterbath	Stovall	HWB115	PRODUCT COULD BE USED.
Cell counter	Nexcelom	Cellometer Auto T4
Two (2) Cell incubators, one at 33 °C/10% CO2 and other at 39 °C/5% CO2	Forma Scientific	3110
Cell culture dishes, PS, 100 mm x 20 mm with vents	Greinier Bio-One	664-160
Cell culture dishes , PS,  60 mm x 15 mm with vents	Greiner Bio-One	628160
Cellstar tissue culture flasks  250 ml	Greiner Bio-One	658-175
Cellstar tissue cultur  flasks 550 ml	Greiner Bio-One	660-175
6 well cell culture plate, with lid-Cellstar	Greiner Bio-One	657-160
Microtest Tissue culture plate, 96 well, flat bottom with lid	Becton Dickinson	353072
Micro-Assay-Plate, Chimmey, 96-well white, clear bottom	Greiner Bio-One	655098
50 ml Polypropylene conical tube with cap Cellstars	Becton Dickinson	352070
15 ml Polypropylene conical tubes with cap-Cellstars	Greiner Bio-One	188-271
PBS pH 7.4 (1x)	Life Technologies	10010-023
Dulbecco’s Modified Eagle’s Medium (DMEM)	Life Technologies	11965-084
Fetal bovine serum (FBS)	Hyclone	SH10073.1
Leibovitz's L-15 Medium, no phenol red	Gibco/Invitrogen	21083-027
Trypsin, 0.25%	Life Technologies	25200-056
TACS MTT Cell Proliferation Assay Kit	Trevigen	4890-25-K
Caspase-Glo 3/7 Assay  kit	Promega	G8091
BrdU Cell Proliferation Assay Kit	Cell Signaling	6813
Non-enzymatic cell dissociation solution	Sigma-Aldrich	C5789
Cell-Tox Green Cytotoxicity Assay Kit	Promega	G8741
FACSAriaIII instrument	BD Biosciences	FACSAriaIII	With 488 nm excitation (blue laser)
Digital Blot Scanner	LI-COR	C-DiGit
Electrophoresis and Blotting Unit	Hoefer	SE300 miniVE
Spectra Max 5 Plate Reader with Soft Max Pro 5.2 Software	Molecular Devices	SpectraMax 5
Patch-clamp amplifier	HEKA	EPC-10
Puller for preparing patch electrodes	Sutter Instruments	P-97