Long-term Time Lapse Imaging of Mouse Cochlear Explants

Summary

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

Abstract

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Introduction

The mammalian cochlea is a highly ordered complex organ. In the mouse, between the emergence of the primitive inner ear from the otic vesicle on day E11 and completion of the developmental program at early postnatal stages, multiple waves of cell signaling and coordinated changes in gene expression take place. Running from base to apex of the cochlear duct is the sound detecting sensory epithelium, or organ of Corti. Development of the organ of Corti is exquisitely controlled such that by the end of development, it will consist of a single row of inner hair cells, three rows of outer hair cells interspersed with five rows of supporting cells (two rows of pillar cells,....

Protocol

Mouse tissue was harvested from Sox2^EGFP-reporter mice¹² maintained and euthanized in accordance with Canadian Council on Animal Care guidelines for the care and use of laboratory animals.

1. Culturing Embryonic Cochleae

1. Supplement Dulbecco’s Modified Eagle Medium (DMEM) by mixing 8.89 ml of DMEM, 1 ml of fetal bovine serum (FBS), 100 μl of 100x N2 supplement, and 10 μl of 10 mg/ml ciprofloxacin. Supplement Hank’s Balanced Salt Solution (HBSS) by mixing 495 ml HBSS with 5 ml of 100% HEPES.
2. Prepare glass bottom culture dishes:
   1. In a laminar flow hood, resus....

Results

Here we show a montage (Figure 1) and a movie (Figure 2) demonstrating how a typical organotypic cochlear explant will grow if plated on E13.5. A Sox2 reporter mouse was used to visualize the prosensory region. The movie illustrates that the cochlea undergoes growth and convergence and extension, the cells in the lateral region of the green Sox2 domain do not seem to divide as the tissue surrounding it expands. This is a characteristic of the organ of Corti; on E13 the .......

Discussion

There are several technical points to consider when cultures are established and in setting up the time-lapse microscope in order for long-term imaging.

We use basement membrane matrix as a substrate for culturing cochlear explants, but the substrate should be matched to the cell type. For example, to image neuronal cultures, it may be better to provide a fibronectin coating. Incubation temperature and gas composition should also be chosen according to tissue type. Choosing the age of the expl.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dulbecco's Modified Eagle media	Gibco	12430	Multiple brands manufacture this
Basement membrane extract	Corning	354230	Matrigel. Alternative similar products are available from other suppliers. http://catalog2.corning.com/Lifesciences/en-US/Shopping/Product.aspx?categoryname=Cell+Culture+and+Bioprocess%28Lifesciences%29|Extracellular+Matrix+Proteins+ECMs+and+Attachment+Factors%28Lifesciences%29|Matrigel+Basement+Membrane+Matrix+%28Lifesciences%29
Fetal bovine serum	Gibco	16000044	Multiple brands manufacture this http://www.lifetechnologies.com/search/global/searchAction.action?query=fbs&resultPage=1&results PerPage=15&autocomplete=
HEPES	Gibco	5630080	Multiple brands manufacture this http://www.lifetechnologies.com/search/global/searchAction.action?query=hepes&resultPage=1&results PerPage=15&autocomplete=
100 x N2 supplement	Gibco	17502-048	Multiple brands manufacture this http://www.lifetechnologies.com/search/global/searchAction.action?query=n2&resultPage=1&results PerPage=15&autocomplete=
Ciprofloxacin	Sigma Aldrich	17850-5G-F	Multiple brands manufacture this http://www.sigmaaldrich.com/catalog/product/fluka/17850?lang=en®ion=CA
Hank's balanced salt solution	Gibco	14170161	Multiple brands manufacture this. Should be refidgerated before use. http://www.lifetechnologies.com/us/en/home/life-science/cell-culture/mammalian-cell-culture/reagents/balanced-salt-solutions/hbss-hanks-balanced-salt-solution.html?s_kwcid=AL!3652!3!26107410508!e!!g!!hbss&ef_id=xoFOglw2s UMAAMU8:20140228185720:s
Fine Forceps	Fine Science tools	11254-20	Size number 5 http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=350&CategoryId=29
Curette	Fine Science Tools	10080-05	size 1 mm  http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=91&CategoryId=118
Insect Pins	Fine Science Tools	26001-35	Must be stainless Steel http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=124
50 mm plastic dishes	Corning/Falcon	351006	Multiple brands manufacture this
charcoal	Sigma Aldrich	05105-250G	Multiple brands manufacture similar items http://www.sigmaaldrich.com/catalog/product/fluka/05105?lang=en®ion=CA
184 silicone elastomer	Dow/Corning	SYLGARD® 184 SILICONE ELASTOMER KIT	Dishes are home made several weeks in advance.  Silicone elastomer can be from any supplier.http://www.dowcorning.com/applications/search/products/Details.aspx?prod=01064291
Glass bottom dishes	MatTek	P35G-0-10-C	The dimensions of the dish are determined by the specifications of the imaging system.  35 mm diameter, 10mm well, number 0 coverslip fits Olympus Vivaview FL. http://glass-bottom-dishes.com/catalog/index.php?main_page=product_info&cPath= 1_4_15&products_id=2
Stereomicroscope	Zeiss	495101-9804-000	Stemi 2000 model. multiple brands manufacture similar items http://microscopy.zeiss.com/microscopy/en_de/products/stereo-zoom-microscopes/stemi-2000.html
Cold Light Source	Zeiss	000000-1063-182	Multiple brands manufacture similar items http://microscopy.zeiss.com/microscopy/en_de/products/microscope-components/lightsources.html
Fluorescent Stereomicroscope	Leica microsystems	Contact Leica microsystems	Leica M165-FC. Multiple brands manufacture similar items http://www.leica-microsystems.com/products/stereo-microscopes-macroscopes/fluorescence/details/product/leica-m165-fc/
Incubator Microscope +imaging software	Olympus	Contact Olympus	Inverted microcope sealed inside a Co2 incubator. Vivaview FL incubator microscope with proprietry Metamorpoh imaging software. http://olympuscanada.com/seg_section/product.asp?product=1055&c=0
Clean bench	Thermo Scientific	51029701	Multiple brands manufacture similar items http://www.thermoscientific.com/en/product/heraguard-eco-clean-bench.html
CO2 incubator	Thermo Scientific	3310	Multiple brands manufacture similar items http://www.thermoscientific.com/en/products/co2-incubators.html
Laminar Flow hood	Thermo Scientific	51026651	Multiple brands manufacture similar items http://www.thermoscientific.com/en/products/biological-safety-cabinets-clean-benches.html