A Unified Methodological Framework for Vestibular Schwannoma Research

Summary

The goal of this protocol is to outline the collection and processing of human surgical samples for multiple downstream applications in vestibular schwannoma and Schwann cell research.

Abstract

Vestibular schwannomas are the most common neoplasms of the cerebellopontine angle, making up 6-8% percent of all intracranial growths. Though these tumors cause sensorineural hearing loss in up to 95% of affected individuals, the molecular mechanisms underlying this hearing loss remain elusive. This article outlines the steps established in our laboratory to facilitate the collection and processing of various primary human tissue samples for downstream research applications integral to the study of vestibular schwannomas. Specifically, this work describes a unified methodological framework for the collection, processing, and culture of Schwann and schwannoma cells from surgical samples. This is integrated with parallel processing steps now considered essential for current research: the collection of tumor and nerve secretions, the preservation of RNA and the extraction of protein from collected tissues, the fixation of tissue for the preparation of sections, and the exposure of primary human cells to adeno-associated viruses for application to gene therapy. Additionally, this work highlights the translabyrinthine surgical approach to collect this tumor as a unique opportunity to obtain human sensory epithelium from the inner ear and perilymph. Tips to improve experimental quality are provided and common pitfalls highlighted.

Introduction

Vestibular schwannomas (VSs) are the most common neoplasms of the cerebellopontine angle, diagnosed in 1 in every 100,000 individuals. Though non-metastatic, these tumors severely affect a patient's quality of life^1,2,3,4,5,6. Affected individuals commonly live with hearing loss, tinnitus, and a feeling of aural fullness. Symptoms become increasingly debilitating as the tumor grows, causing balance problems, facial paralysis, and impairment of other cranial nerve funct....

Protocol

Written informed consent for the collection of all samples was obtained prior to surgery, and the experiments were carried out according to the Code of Ethics of the World Medical Association (Declaration of Helsinki). All sections of the study protocol were approved by the Institutional Review Board of Massachusetts Eye and Ear and Massachusetts General Hospital.

NOTE: Sections 1-7 below are designed to be performed sequentially upon the receipt of a primary human VS or GAN sample from the operating room. Processing should always begin with section 1. Then, as a rule, RNA should be preserved first (section 2), followed by the preparation o....

Results

Primary human VS cells in culture, as established in section 5, can be treated as informative models for disease-associated processes in many downstream research applications (Figure 1). Healthy Schwann cells cultured in section 6 provide a direct and instructive point of comparison. As outlined below, extensive data from VSs and GANs processed according to this unified methodological framework are available in multiple articles previously published

Discussion

This manuscript describes a unified methodological framework for VS research, outlining the simultaneous processing of human VS and GAN specimens for downstream research applications. As VS research enters the age of precision medicine, preparing the same sample in forms capable of answering numerous research questions will enable the discovery of molecular, cellular, genetic, and proteomic insights specific to individual patients.

The purity of human Schwann cell and VS cultures were thorough.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BioCoat Poly-D-Lysine/Laminin 12 mm #1 German Glass Coverslip	Corning	354087	Or prepare coverslips with Corning Laminin (CB-40232) and Cultrex Poly-L-Lysine (3438-100-01)
CELLSTAR 15 mL Centrifuge Tubes, Conical bottom, Graduation, Sterile	Greiner Bio-One	188161
CELLSTAR 50 mL Centrifuge Tubes, Conical bottom, Graduation, Sterile	Greiner Bio-One	227261
CELLSTAR Cell Culture Dish, 60 mm	Greiner Bio-One	628160
Collagenase from Clostridium histolyticum, Sterile-filtered	Sigma-Aldrich	C1639
Costar 24 Well Clear TC-Treated Multiple Well Plates, Sterile	Corning	3526
DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride)	Thermo Fisher Scientific	D1306
DMEM, high glucose, pyruvate, no glutamine, 500 mL	Thermo Fisher Scientific	10313-039
DMEM/F-12, 500 mL	Thermo Fisher Scientific	11320-033
Dumont #3 Forceps, Dumoxel	Fine Science Tools	11231-30	Autoclave prior to use
Dumont #5 Forceps, Standard tip, Inox	Fine Science Tools	11251-20	Autoclave prior to use
Fetal Bovine Serum, qualified, USDA-approved regions, 500 mL	Thermo Fisher Scientific	10437-028	Aliquot in 50 mL tubes and store in -20°C freezer
Hyaluronidase from Bovine Testes, Type I-S, Lyophilized Powder	Sigma-Aldrich	H3506
Millex-GP Syringe Filter Unit, 0.22 µm, polyethersulfone, 33 mm, sterile	EMD Millipore	SLGP033RS
Paraformaldehyde, Reagent Grade, Crystalline	Sigma-Aldrich	P6148	Prior to use: Establish Standard Operating Procedures based on protocols available online
PBS, pH 7.4, 500 mL	Thermo Fisher Scientific	10010-023	Autoclave prior to use
Penicillin-Streptomycin (10,000 U/mL), 100 mL	Thermo Fisher Scientific	15140-122
PhosSTOP Phosphatase Inhibitor Tablets	Roche	04906845001
Pierce Protease Inhibitor Tablets	Thermo Fisher Scientific	88666
Pipettes and pipette tips, 5/10/25 mL	Variable	Variable
Plastic Homogenization Pestle for 1.5/2.0 mL Microtubes	E&K Scientific	EK-10539
PrecisionGlide Needles, 27 G x 1 1/2 in	BD	301629
RIPA Buffer	Boston BioProducts	BP-115
RNAlater (RNA stabilization solution)	Thermo Fisher Scientific	AM7021
Safe-Lock Microcentrifuge Tubes, Polypropylene, 0.5 mL	Eppendorf	022363719	Autoclave prior to use
Safe-Lock Microcentrifuge Tubes, Polypropylene, 1.5 mL	Eppendorf	022363204	Autoclave prior to use
Saline - 0.9% Sodium Chloride Injection, bacteriostatic, 20 mL	Hospira	0409-1966-05
Scalpel Blades - #15	Fine Science Tools	10015-00
Schuknecht Suction Tube 24 gauge	Bausch + Lomb	N1698 42	Useful for the surgical approach (in addition to common otologic surgical instruments) and e.g. a blue surgical marker
Specimen Container, OR sterile, 4OZ	Medline	DYND30331H
Stemi 2000-C Stereo Microscope	Zeiss	000000-1106-133
Syringe/Needle Combination, Luer-Lok Tip, 5 mL, 22 G x 1 in.	BD	309630
Tuberculin Syringe Only, Slip tip, 1 mL	BD	309659
Tuberculin Syringe Only, Slip tip, 3 mL	BD	309656
Ultrasonic homogenizer, 4710 Series, CV18 probe	Cole-Parmer	CP25013