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.
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.
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 life1,2,3,4,5,6. 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 functions. Life-threatening complications due to brainstem compression may also ensue7.
Management options for VS are essentially limited to watchful waiting for static tumors and stereotactic radiation therapy or surgical resection for growing tumors8. The surgical removal of these tumors in research-affiliated hospitals presents the opportunity to acquire and analyze fresh tumor tissue collected during patient surgeries. One specific surgical approach to VS, the translabyrinthine resection, can even offer access to valuable human sensory epithelium from the inner ear and perilymph.
Because VSs arise from a peripheral sensory nerve (i.e., the vestibular nerve), it is important to compare VS-associated observations with those derived from an appropriate control nerve, such as the human great auricular nerve (GAN). Healthy GANs are regularly sacrificed during parotidectomies or neck dissections and can be used as robust models for healthy Schwann cell physiology9.
Because there are no FDA-approved drugs for the treatment or prevention of sporadic VS, it is imperative that researchers elucidate the underlying molecular mechanisms of the disease to identify therapeutic targets. Proteins that have been shown to play a role in VS pathogenesis include merlin, vascular endothelial growth factor (VEGF), cyclooxygenase 2 (COX-2), nuclear factor kappa B (NF-κB), tumor necrosis factor alpha (TNF-alpha), epidermal growth factor receptor (EGFR), and related signaling molecules10,11,12,13,14,15,16,17.
Recent advances have expanded and improved protocols for the collection, processing, culture, and downstream investigation of primary human vestibular schwannomas and healthy nerve tissues18,19. However, most existing protocols are designed to accommodate the preparation of such tissues for a single downstream research application (i.e., cell culture alone). This article presents a unified methodological framework for the simultaneous processing of a single primary human VS or GAN sample for multiple downstream applications: cell type-specific culture, protein extraction, RNA preservation, tumor secretion collection, and tissue fixation. This work also details the surgical collection and processing of human cerebrospinal fluid (CSF) and perilymph during translabyrinthine VS resection, as these closely related tissues may serve as important sources of biomarkers for VS. Finally, this protocol presents steps for the viral transduction of primary human VS cells in culture as a novel application of this tissue for use in gene therapy.
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 of tissue for the collection of secretions (section 3) and protein extraction (section 4). Tissue to be cultured (section 5 for VS, section 6 for GAN) can sit in supplemented culture medium while sections 2, 3, and 4 are performed. The fixation of tissue for sectioning (section 7) is very short and can be performed during any centrifugation step. Section 8 (perilymph processing) and section 9 (CSF processing) can be carried out upon the receipt of perilymph or CSF from the operating room during a translabyrinthine VS resection. Section 10 (viral transduction) can be performed on growing VS or Schwann cells in culture.
1. Preparation for and Receipt of a Surgical Sample
NOTE: The following steps are to be performed in a sterile environment, such as a tissue culture hood or laminar flow hood. Familiarity with basic sterile technique is expected.
2. RNA Preservation of VS and GAN Tissue (Also Valid for Human Sensory Epithelium)
3. Collection of the VS and GAN Secretions
4. Protein Extraction from VS or GAN Tissue
5. Primary Human VS Culture
6. Primary Human GAN Culture
7. VS & GAN Tissue Fixation
8. Collection and Storage of Human Perilymph during Translabyrinthine VS Resection
9. Collection and Storage of Human CSF
10. Viral transduction experiments for primary VS cells
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 published12,13,14,15,27.
The successful transduction of primary VS cells with adeno-associated viruses for gene therapy is presented here for the first time (Figure 2). Primary VS cells were transduced in culture with GFP-expressing Anc80, a predicted ancestor of adeno-associated viruses28. Anc80 has been shown to be a maximally efficient vector for gene transfer in murine cochlear tissues in vitro and in vivo27. The effective transduction of primary human cells with this vector carries important implications for future forays into gene therapy for VS.
Figure 1: Bright-field Images of Primary Human Vestibular Schwannoma Cultures.
Typical patterns in the organization of VS cells in culture can be identified. Scale bar = 200 µm. Please click here to view a larger version of this figure.
Figure 2: Representative Immunofluorescent Image of Primary Human Vestibular Schwannoma Cultures after Exposure to GFP-expressing Anc80 for 48 h at a Multiplicity of Infection (MOI) of 250,000.
Green = GFP; red = phalloidin/F-actin (stains the cytoskeleton); blue = DAPI (stains the nucleus). Scale bars = 50 µm (A) and 100 µm (B). Please click here to view a larger version of this figure.
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 thoroughly assessed over time by means of immunocytochemistry, using the ratio of cells positive for the S100 marker to those positive for the DAPI nuclear stain19. Accordingly, it is recommended to perform experiments on primary human Schwann cell cultures within the first two weeks after plating the cells, as the purity of these cells is the highest during this period; afterwards, fibroblast-like cells begin to predominate. Though VS cell cultures are also maximally pure at two weeks in culture, VS cells lack contact-mediated inhibition and will continue to proliferate at later stages. Additionally, a direct microarray comparison of protein expression from VS tissue (section 4) and VS cells in culture from the same tumors (section 5) successfully showed that VS cultures demonstrate a satisfactorily high level of biological similarity to their respective parent tumors19. The successful quantification of proteins of interest can be performed via Western blot of protein lysates generated in section 4 and the quantification of RNA transcripts via qRT-PCR of RNA extracted from tissues preserved with RNA stabilization solution (section 2)12,13,14.
VS and GAN cells in culture can be exposed to chemically active substances, such as nonsteroidal anti-inflammatory drugs, small interfering RNAs (siRNAs), or natural organic compounds to elucidate disease-specific molecular mechanisms or to test a therapeutic target13,14,29. Compounds of interest can be directly added to cells in culture after appropriate dilutions in regular supplemented culture medium. To assess the effect of such substances on important aspects of cellular physiology, bromodeoxyuridine (BrdU) labeling for proliferation, terminal deoxynucleotidyl tranferase dUTP nick end labeling (TUNEL) for apoptosis, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction for metabolic viability are reliable assays that can be used with confidence on these cells19. Culture medium from treated cells can also be carefully aspirated, stored at -20 °C, and tested to quantify relative levels of secreted substances, such as prostaglandin E2, the secretion of which can be affected by drug treatment13. Additionally, tissue fixation (section 7) and subsequent embedding in OCT or paraffin provides a robust substrate for immunofluorescence assays13,14.
The tumor- or nerve-conditioned medium generated in section 3 provides a simple and effective way to assess VS-secreted soluble molecules and to extract extracellular vesicles. Cytokine arrays or ELISAs can be carried out on these secretions, as indicated in the manufacturers' protocols, outlined in two published studies9,12. Extracellular vesicles can be purified from these secretions using ultracentrifugation, as detailed in a previous publication30. Alternatively, the secretions themselves can be used as patient-specific reagents for downstream research applications. For example, in an experiment that revealed a novel mechanism underlying VS-associated sensorineural hearing loss, secretions were collected from the VS tissue of patients with poor hearing and VS patients with good hearing after incubation in DMEM for 72 h (section 3). These tumor secretions were then applied to murine cochlear explants, and it was revealed that tumor secretions from VS patients with poor hearing caused more damage to cochlear explants than secretions from VS patients with good hearing15. Importantly, secretions collected at time points before 72 h did not result in substantial damage.
Human perilymph collected from patients undergoing translabyrinthine VS resection (section 8) represents an exciting new avenue for the discovery of VS biomarkers. Specimens collected according to section 8 were analyzed via liquid chromatography-tandem mass-spectrometry (LC-MS/MS) to map the proteome of human perilymph, and 15 candidate biomarkers of VS were successfully identified31. A similar analysis could be possible using human CSF collected from VS patients (section 9).
A significant consideration in VS research is the selection of appropriate control tissue. Previous studies have used the cochlear nerve, the vestibular nerve, and unrelated peripheral nerves (such as the sciatic nerve), to variable effect22,32. However, several reasons lead to the support of the use of GANs as maximally relevant controls15. Similar to the vestibular nerve, the GAN is a peripheral, sensory nerve with axons ensheathed by Schwann cells. Importantly, a literature search reveals that schwannomas have never been found to develop from the GAN, making neoplastic changes in this tissue improbable. Additionally, GANs are regularly sacrificed when accessing structures of the deeper neck, giving hospital-based researchers easy access to healthy specimens during routine neck dissections and parotidectomies. Although the cochlear nerve is often sacrificed during VS surgery and may be readily available, its close proximity to the vestibular nerves risks the sharing of the same microenvironment, which may demonstrate pre-tumorous molecular changes33. Other laboratories have also successfully utilized GANs as an adequate control for VS research, and it is recommended to continue this practice20,21,22,34.
One critical but often overlooked step within the cell type-specific culture protocols (sections 5 and 6) is the proper removal of non-viable tissue and blood vessels. Removal of these non-tumor tissues is crucial to generate robust cultures of maximal purity. Furthermore, when plating cell-containing culture medium onto coverslips, it is important to pay close attention to the amount of tissue transferred to each well. Achieving an even, mildly dense distribution of cells containing a few "chunks" of denser tissue in each well is particularly important for Schwann cells, which require a sufficient number of adherent cells to thrive. Coating the coverslips with poly-D-lysine and laminin also allows for the efficient growth of cells. Cells proliferate more robustly when the coverslips are coated in the laboratory, as compared to commercially available pre-coated products.
To ensure a high-quality protein and RNA extraction, verify that the tissue remains at temperatures below 4 °C throughout sections 2 and 4. Additionally, since the literature regarding the analysis of VS secretions is scarce (section 3), new projects might require further innovations and different lengths of incubation.
As methods to address VS pathobiology continue to advance, the use of this streamlined combination of fundamental techniques will allow the maximum amount of information to be gleaned from a single human sample. The informed simultaneous processing of VS and GAN tissues will prove integral to the generation of effective pharmacological and genetic therapies against this debilitating tumor.
The authors have nothing to disclose.
This work was supported by the National Institute of Deafness and Other Communication Disorders grants R01DC015824 (K.M.S.) and T32DC00038 (supporting J.E.S and S.D.), the Department of Defense grant W81XWH-14-1-0091 (K.M.S.), the Bertarelli Foundation (K.M.S.), the Nancy Sayles Day Foundation (K.M.S.), the Lauer Tinnitus Research Center (K.M.S.), and the Barnes Foundation (K.M.S.).
BioCoat Poly-D-Lysine/Laminin 12mm #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 1.5/2.0ml 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 |