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 alternative therapies has driven investigation into the unique molecular biology of VSs in hopes of developing novel treatments^3,4.

Cell culture allows for rapid, facile, and in-depth analysis of molecular and cellular behavior and screening of potential therapeutic compounds. 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 oncogenes⁵. This oncogenic transduction introduced significant molecular and phenotypic alterations to the cells, which limit their use as a model for human schwannoma tumors. SC cultures derived from transgenic mouse lines that lack a functional NF2 gene represent another alternative to investigate NF2-dependent SC tumorigenesis in vitro. These cultures however fail to recapitulate the heterogeneous nature of human VSs or account for human specific behavior. Most previous VS culture techniques required relatively long processing times and complicated selective culture techniques^6-8. Here we present a simple, reproducible protocol for primary VS cell culture with complete processing in under 3 hr, with 95% tumor cell purity as determined by immunostaining.

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 culture hoods. Add enough laminin solution (10 µg/ml in Ca²⁺/Mg²⁺-free HBSS [HBSS-/-]) to completely cover the culture plates, replace lids, then either 1) place in cool 4 °C refrigerator overnight, or 2) place in 37 °C incubator for at least 2 hr. If plates will be stored overnight or longer, wrap the plates in plastic wrap to prevent evaporation/desiccation.

2. Setup the Day of Tumor Harvest

1. Prepare Culture Media: Media is made fresh on day of tumor harvest and processing, stored at 4 °C, and warmed to 37 °C in incubator immediately prior to media additions/changes.
2. Schwannoma culture media is high-glucose (4.5 mg/ml) DMEM (with phenol red) with 0.1 mg/ml penicillin and 0.1 mg/ml streptomycin; N2 media supplement final dilution 1:100; Bovine Insulin 5 µg/ml; Fetal Bovine Serum to 10% total volume. Filter all media through 0.22 μm filter.
3. Prepare Specimen Transport Cooler: Send an ice filled cooler with 1-2 (depending on amount of tumor to be harvested) sterile 50 ml conical tubes with 25 ml HBSS with Ca²⁺/Mg²⁺ (HBSS+/+) to the operating room for tumor sample transfer and transport.
4. Prepare individual tumor dissociation tubes – fill sterile 2 ml round bottom tubes with 1 ml of HBSS+/+ and place on ice. (Tubes will be used for sharp dissection/dissociation of tumor samples).
5. For dissociation, prepare approximately one dissociation tube for every 0.5 cm³ of harvested tissue; for example, a tumor specimen measuring 1.0 x 1.0 x 1.0 cm (1 cm³) will require two HBSS+/+ dissociation tubes.
6. Ultraviolet light sterilization: Place tissue scissors, small forceps, scalpel handle, 1x normal Petri dish, P1000 pipette, and pipette tips in a sterile culture hood, and leave under ultraviolet light for ~15 min prior to arrival and processing of tumor tissue.

3. Specimen Isolation and Transport

1. Isolate tumor specimens by surgical resection.
2. Immediately place tumor specimens into the ice-cold HBSS+/+ as quickly as possible after removal from the patient. Once all tissue samples are collected, transport samples (on ice) from the operative theater to the lab for further processing.

4. Tissue Dissociation and Trituration

1. Following standard sterile technique in a laminar flow hood, bring conical tube with tumor specimen into the culture hood, and wash samples by inverting tube and tumor 50x. Allow fragments to fall to the bottom of the tube, and then remove HBSS+/+. Refill with 30 ml HBSS+/+, and agitate/invert 50x again. Repeat inversion/media removal for a total of 4x.
2. Re-suspend tumor fragments in 30 ml HBSS+/+ for the last time, then pour mixture into a sterile100 mm petri dish, and separate tumor into 0.5 cm³ groupings. If larger tumor fragments are encountered, use the tissue scissors or scalpel (#11 or #15 blade) to cut into smaller pieces.
3. Use the forceps to place the 0.5 cm³ tissue groups into the individual HBSS+/+ filled, 2 ml round bottom conical tubes, all on ice. Use the tissue scissors to mince tumor fragments into sub-1 mm fragments –the suspension should have a ‘snowglobe’ appearance (Figure 1). Mince the tumor only until the majority of the fragments are sub-1 mm; do not ‘over-mince’ the tumor samples. Place completely minced tissue tube back on ice, and repeat with all additional specimen tubes.
4. Transfer all fragmented tumor samples into a single 15 ml conical tube. A P1000 pipette with a cut/modified sterile tip works well for this step. Use extra HBSS+/+ to flush/rinse the 2 ml dissociation tubes to make sure all tumor sample has been collected in 15 ml tube. Spin down mixture in centrifuge at 23 °C, at 73 x g for 5 min.
5. Suction off HBSS+/+, leaving the cell pellet. Re-suspend fragments in 1:1 mixture of 0.25% trypsin:0.2% collagenase (dissolved in HBSS-/-) – add just enough to keep tumor fragments in tight suspension. Replace screw-top, and place in 37 °C incubator for 30 min. Agitate cell mixture at least once during incubation to keep fragments in suspension. Place the Schwannoma culture media into the incubator to warm it at this time as well.
6. After incubation, add 100 µl FBS to each tube to inactivate the trypsin, and then centrifuge sample at 23 °C, 73 x g for 5 min. Using a pipette, carefully suction off as much supernatant as possible without disturbing cell pellet. Add the warmed culture media (N2/insulin/5% FBS) to the tumor fragments to a final ratio of 1:2 tumor fragments: culture media (for example, if there is 1 ml of tumor fragments, add in 2 ml of warmed media).
7. Prepare for tumor trituration by cutting the tip off of a P1000 pipette tip to a diameter of approximately 1-2 mm. Slowly triturate the cell suspension using progressively smaller and smaller diameter pipette tips, until you can easily pipette the solution through an unaltered P1000 pipette tip. Immediately move to a smaller tip once you can easily pipette the tumor solution through the tip – do not over triturate the sample. If a single larger piece of tissue blocks aspiration during trituration, tapping the pipette tip on the bottom of the conical tube often allows continued trituration.

5. Cell Plating

1. One 0.5 mm³ fragment of tissue is enough to seed 1 x 100 mm dish, or 4 x 8-well/4-well slides. Just prior to adding the media/cell mixture, remove the laminin solution from the culture plates but do not let the plates dry.
2. For each 100 mm dish, dissolve the tumor fragment in 10 ml warmed culture media.
3. For each well of a 4-well slide, use 750 μl warmed culture media (3 ml for the whole slide).
4. For each well of an 8-well slide, use 400 μl warmed culture media (3.2 ml for the whole slide).
5. Incubate the cultures in 37 °C, 100% humidity, 6% CO₂. Leave the cultures alone for at least 36 hours, and then change the media if the solution is yellowed (acidic). Otherwise change the media at 72 hr, then every 2-3 days or when the media become acidic. If the media becomes acidic daily for several days in a row, this is generally an indication of potential bacterial or yeast contamination.

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-adhere...

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

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