Primary Culture of Human Vestibular Schwannomas

Summary

Vestibular Schwannomas (VSs) are non-malignant tumors of Schwann cell (SC) origin, associated with mutations in the NF2 tumor suppressor gene. We report a reproducible, efficient protocol for primary human VS cell culture that allows for molecular and cellular experimental manipulation and analysis and recapitulates the heterogeneous nature of human disease.

Abstract

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in either sporadic or familial (neurofibromatosis type 2, NF2) forms, both associated with inactivating defects in the NF2 tumor suppressor gene. Treatment for VSs is generally surgical resection or radiosurgery, however the morbidity of such procedures has driven investigations into less invasive treatments. Historically, lack of access to fresh tissue specimens and the fact that schwannoma cells are not immortalized have significantly hampered the use of primary cultures for investigation of schwannoma tumorigenesis. To overcome the limited supply of primary cultures, the immortalized HEI193 VS cell line was generated by transduction with HPV E6 and E7 oncogenes. This oncogenic transduction introduced significant molecular and phenotypic alterations to the cells, which limit their use as a model for human schwannoma tumors. We therefore illustrate a simplified, reproducible protocol for culture of primary human VS cells. This easily mastered technique allows for molecular and cellular investigations that more accurately recapitulate the complexity of VS disease.

Introduction

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms^1-3. VSs occur in either sporadic or familial (neurofibromatosis type 2, NF2) forms, both associated with inactivating defects in the NF2 tumor suppressor gene. Treatment for VSs is generally surgical resection or radiosurgery, however the morbidity of such procedures includes deafness, facial neuropathies, spinal fluid leak, imbalance, and tumor regrowth¹. Additionally not all patients are acceptable surgical or radiation candidates. Such significant morbidity as well as a lack of alternati....

Protocol

Ethics Statement: use of the human tumor specimens in this protocol was approved by the University of Iowa Institutional Review Board (IRB).

1. Setup the Day Before Tumor Harvest

1. Coat Cell Culture Plates: In a sterile culture hood, add enough poly-L-ornithine (0.01% solution) to cover the bottom of a polystyrene/plastic culture well/dish, replace the lids, and let sit at room temperature for at least 2 hr. Glass slides or coverslips can be substituted for polystyrene for experiments requiring imaging.
2. After 2 hr, remove the poly-L-ornithine solution with suction and allow the plates to dry completely in a sterile cultu....

Results

Correct tumor fragmentation is essential for optimal seeding and outgrowth in the tissue culture dishes (Figure 1). Identification of schwannoma cells during the first days after plating is often difficult, due to obscuring amounts of cellular debris and red blood cells. By the 4^th or 5^th day, cell extensions near adherent tumor fragments will create recognizable ‘lacing’ patterns among the debris. Subsequent media changes generally remove the majority of non-adherent cells by the s.......

Discussion

History of in vitro Schwannoma Cultures

Efforts to establish in vitro schwannoma cultures began just a few decades after the initial acoustic neuroma open surgical resection occurred in 1894¹². The first recorded cultures were by Kredel in the 1920s, who unsuccessfully attempted to grow minced tumor by “hanging drop method”¹². Later, Drs. Murray and Stout published their in vitro culture experience with neurilem.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Poly-L-Ornithine, 0.01% Solution	Sigma	P4957	Cell Culture Surface Treatment
Laminin mouse protein	Gibco	23017-015 / L2020	Cell Culture Surface Treatment
0.2% Collagenase (dissolved in HBSS-/-)	Sigma	C2674	Dissociation Reagent
0.25% Trypsin	Gibco	25200-056	Dissociation Reagent
TPS 100 mm round culture dish	Midwest Scientific	TP93100
Permanox 4-well slide	Fisher Scientific	1256521
Permanox 8-well slide	Fisher Scientific	1256522
Suction filter flask	Midwest Scientific	TP99500
15 ml Conical tube	Midwest Scientific	TP91015
50 ml Conical tube	Midwest Scientific	TP91050
2 ml Round bottom tube	USA Scientific	1620-2700
Small scissors	FST	14058-11
Small forceps	FST	11251-20
Scalpel handle	FST	10004-13
#11 Scalpel blade	Roboz	RS-9801-11
Non-tissue culture 100 mm round Petri dish	Fisher Scientific	50-820-904
P1000 Pipetteman	Bioexpress	P3963-1000
Serological pipetteman	Bioexpress	R3073-2P
Sterile, non-filtered P1000 pipette tips	Midwest Scientific	TD1250R
Insulated ice cooler
Culture hood	Baker
Centrifuge	Eppendorf -5810R
Hanks Balanced Salt Solution (HBSS)+/+ (w/ Ca2+, Mg2+)	Gibco	24020-117	Schwannoma Culture and Media Components
Hanks Balanced Salt Solution (HBSS)-/- (w/o Ca2+, Mg2+)	Gibco	14170-112	Schwannoma Culture and Media Components
Dulbecco’s Modified Eagle Medium (DMEM), High Glucose, w/ Phenol Red	Gibco	11965-092	Schwannoma Culture and Media Components
N2 supplement	Gibco	17502-048	Schwannoma Culture and Media Components
Fetal Bovine Serum	Gibco	26140-079	Schwannoma Culture and Media Components
Penicillin/streptomycin	Gibco	15140-163	Schwannoma Culture and Media Components
Bovine insulin (1 mg/ml 200x)	Sigma	I6634	Schwannoma Culture and Media Components