A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering

Summary

The goal of this protocol is to demonstrate an effective method to decellularize and decalcify mouse cochleae for utilization as scaffolds for tissue engineering applications.

Abstract

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current regenerative medicine strategies have focused on transplanting stem cells or genetic manipulation of surrounding support cells in the inner ear to encourage replacement of damaged stem cells to correct hearing loss. Yet, the extracellular matrix (ECM) may play a vital role in inducing and maintaining function of hair cells, and has not been well investigated. Using the cochlear ECM as a scaffold to grow adult stem cells may provide unique insights into how the composition and architecture of the extracellular environment aids cells in sustaining hearing function. Here we present a method for isolating and decellularizing cochleae from mice to use as scaffolds accepting perfused adult stem cells. In the current protocol, cochleae are isolated from euthanized mice, decellularized, and decalcified. Afterward, human Wharton's jelly cells (hWJCs) that were isolated from the umbilical cord were carefully perfused into each cochlea. The cochleae were used as bioreactors, and cells were cultured for 30 days before undergoing processing for analysis. Decellularized cochleae retained identifiable extracellular structures, but did not reveal the presence of cells or noticeable fragments of DNA. Cells perfused into the cochlea invaded most of the interior and exterior of the cochlea and grew without incident over a duration of 30 days. Thus, the current method can be used to study how cochlear ECM affects cell development and behavior.

Introduction

The cochlea is an intricate spiral structure found in the temporal bone. It is composed of an outer bony labyrinth and a concentric, inner membranous labyrinth¹. The membranous labyrinth consists of three fluid spaces: Scala vestibuli, Scala media, and Scala tympani¹. The scala media houses the sensory epithelium, which is composed of a multitude of cell types, but the sensory hair cells (HC), which transduce mechanical energy in sound waves to nerve impulses², are of particular interest. Exposure to acoustic trauma^3,4,

Protocol

All procedures, including animal euthanasia, were conducted according to the approved Institutional Animal Care and Use Committee (IACUC) protocol (ACUP #2014-2234) at the University of Kansas Medical Center (KUMC).

NOTE: HWJCs were isolated from human umbilical cords that were donated by patients that provided informed consent and specimens were used in accordance with the protocols approved by the University of Kansas Human Subjects Committee (KU-IRB #15402).

1. Temporal Bone Harvest and Cochlea Isolation

1. Decapitate an appropriately euthanized, 15-week-old mouse by cutting between the skull and firs....

Results

Using the methods presented here, successful decellularization of cochleae was assessed by examining the presence or absence of DNA through 4',6-diamidino-2-phenylindole (DAPI) staining. Cochleae were considered fully decellularized if DNA was not identified within the decellularized cochlea. A native cochlea from a previous experiment that did not undergo decellularization or decalcification was used as a positive control to illustrate the structures and cells traditionally found in a C5.......

Discussion

We have successfully demonstrated that native cochlear cells can be removed from the cochlea via a decellularization process, which allows for the use of the cochlea as an intricate, three-dimensional tissue scaffold. Santi et al.¹⁵ developed the initial method for decellularizing cochleae, and have accurately estimated the volumes of many cochlear structures through with the aid of light sheet microscopy²³. Such early work served as a strong basis for the tissue e.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Allegra X-14R Centrifuge	Beckman-Coulter	B08861
Intramedic Semi-Rigid Tubing	Becton Dickinson	427401
New Brunswick Innova 2000 Orbital Shaler	Eppendorf	M1190-0002
Surgical Scissors	Fine Science Tools	14060-10
Fine Forceps	Fine Science Tools	11370-40
Ultra-Fine Forceps	Fine Science Tools	18155-13
50-mL Conical Tubes	Fisher Scientific	12565271
Petri Dish	Fisher Scientific	FB087579B
U-100 Insulin Syringe	Fisher Scientific	14-829-1B
Scintillation Vial	Fisher Scientific	03-341-73
Rotator	Fisher Scientific	88-861-049
Transfer Pipette	Fisher Scientific	22-170-404
Razor Blade	Fisher Scientific	12-640
Antibiotic-Antimycotic	Fisher Scientific	15-240-062
Penicillin-Streptomycin	Fisher Scientific	15-140-122
24-Well Plate	Fisher Scientific	07-200-84
SuperFrost PLUS Glass Microscope Slides	Fisher Scientific	12-550-15
Transfer Pipette	Fisher Scientific	22-170-404
ProLong Gold Antifade Mountant with DAPI	Fisher Scientific	P36935
Clear-Rite 3	Fisher Scientific	22-046341
Thermo Scientific Forma Series II 3110 Water-Jacekted CO2 Incubator	Fisher Scientific	13-998-078
Mesenchymal Stem Cell Growth Medium	Lonza	PT-3001
Trypsin-EDTA	Lonza	CC-3232
TPP T-75 Culture Flask	MidSci	TP90076
TPP T-150 Culture Flask	MidSci	TP90151
TPP T-300 Culture Flask	MidSci	TP90301
Dissection Microscope	Nikon Instruments	SMZ800
Nikon Eclipse Ts2R-FL Inverted Microscope	Nikon Instruments	MFA51010
NuAire Class II, Type A2 Biosafety Cabinet	NuAire	NU-425-600
1X PBS	Sigma-Aldrich	P5368-10PAK
1% SDS Solution	Sigma-Aldrich	436143-100G
10% EDTA	Sigma-Aldrich	E9884-100G