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.
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.
Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms1-3. 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 treatments3,4.
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 techniques6-8. 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.
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
2. Setup the Day of Tumor Harvest
3. Specimen Isolation and Transport
4. Tissue Dissociation and Trituration
5. Cell Plating
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 4th or 5th 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 second week. Using this method, 90% confluence can be expected between days 7-14, depending on the initial vitality of the tumor fragment prior to mincing and plating density (Figure 2). As described previously, schwannoma cultures appear to grow outward from small fragments of adherent tumor fragments9,10. Such outgrowth is detectable by the end of the first week. Figure 2 shows brightfield imaging of schwannoma cell outgrowth from a single tumor fragment, outlined in red in the lower right corner. Figure 3 illustrates the ‘bridging’ outgrowth that occurs between two different tumor fragments, in culture wells immunostained with polyclonal rabbit anti-S100 antibody. We routinely immunostain cultures with anti-S100 or anti-p75NTR antibodies to verify the purity of cultures and positively identify schwannoma cells for analysis (Figure 4). With these methods over >95% of the cells in these cultures represent schwannoma cells. Schwannoma cells also label with glial fibrillary acidic protein (GFAP) and ErbB2, among other markers11.
Figure 1. Comparison of the same tumor sample both before (A) and after (B) mincing. Sample prepared in 1.5 ml HBSS+/+ with Phenol Red in 2.0 ml round bottom conical tube.
Figure 2. Typical appearance of cultures on brightfield microscopy. Spindled schwannoma cell outgrowth radiates from tumor fragment, image taken on culture day 10. Scale bar = 100 µm.
Figure 3. Immunostaining of primary vestibular schwannoma cultures with anti-S100 antibodies demonstrating typical culture and cell morphologies. A) Low power view illustrating outgrowth from plated tumor fragments, culture day 14. Scale bar = 200 µm. B) Higher power view of schwannoma cells with spindled morphology, culture day 14. Scale bar = 100 µm.
Figure 4. Immunostaining of primary vestibular schwannoma cultures to confirm culture purity and cell identity. A) Cultures immunostained with anti-S100 antibodies. Nuclei are labeled with DAPI. Scale bar = 100 µm. B) Cultures immunostained with anti-p75NTR antibodies. Nuclei are labeled with DAPI. Scale bar = 100 µm.
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 neurilemomas grown on clotted human plasma. Interestingly, this technique revealed that Antoni A and Antoni B specific fragments grew differently, with unique cellular morphology and differential rates of agar liquefaction13. Cravioto and Lockwood published further observations of acoustic neuroma cultures in avian clots on coverglass slips and roller tubes. They were also the first to use Rose chambers for time-lapse imaging of tumor growth14. Publications in the 1970s investigated growth differences between typical monolayer culture and ‘en bloc’ growth of larger tumor fragments10. Baur combined the ‘en bloc’ and monolayer approaches in her publication, which also demonstrated the mitogenic effects of laminin-coated culture plates9. Most subsequent published protocols, as our own, utilize a similar protocol of seeding minced tumor fragments onto a culture surface treated with a charged amino acid (poly-L-ornithine, poly-L-lysine) and laminin preparation8. We have used this same method to culture schwannomas arising from the facial, trigeminal, vagus, and spinal nerves with similar results.
Benefits of Vestibular Schwannoma Primary Cultures
Studies of human schwannoma tumorigenesis often use homogenous immortalized cell lines and transgenic mice models. Such tools are beneficial but do not accurately reflect the genotypes, phenotypes, and complexity of human disease. By contrast, primary cultures likely provide a more realistic model. They more faithfully recapitulate the heterogeneity of genomic and molecular status associated with human tumors15.
Purity of Schwannoma Cultures
A challenge of primary tissue culture is maintaining target cell purity. Schwannomas overwhelmingly consist of neoplastic Schwann cells6, however cultures are not immune from fibroblast contamination. Historically, methods for optimizing schwannoma cell purity fall into three categories: 1) selection by chemically defined medium and specific growth factors; 2) immunopurification (e.g., with magnetic bead cell sorting7), 3) a combination of both the preceding methods. In our experience fibroblasts or other non-schwannoma cells generally comprise less that 5% of cells in the culture, regardless of patient characteristics (age, sex), sporadic or NF2-associated tumors, primary or secondary tumor resection, or cystic vs solid tumor types. If fibroblasts appear to be taking over the culture, removal of the serum for 1-2 media changes will significantly reduce this population without adversely affecting schwannoma cell survival. Pre-treating cell culture surfaces with laminin also facilitates schwannoma cell growth9. Minimal passaging of the cultures also helps to decrease unwanted cellular contamination – cryostorage is best completed within the first two culture passages, and culture experimentation is not recommended beyond passage 4-5. We find optimal results when culture experimentation is completed by passage 1-2 with non-cryostored cells.
We use both immunostaining (anti-S100, DAPI) and microscopic appearance of cellular and nuclear morphology as quality control measures in our schwannoma cultures. Occasionally it is helpful to immunostain a subset of cultures for monocyte (CD68) or fibroblast specific markers to further verify culture purity9. During experimental analyses of cell behavior (e.g., proliferation or cell death), we always immunostain with anti-S100 or anti-p75NTR antibodies to verify that the findings are schwannoma cell specific.
Culture morphology and growth patterns
Schwannoma tumor cultures typically display unique growth patterns reminiscent of Antoni A (high cell density, tightly organized, indications of Verocay body formations) and rarely of Antoni B (comparatively sparse nuclei, abundant cellular processes, less organization) patterns seen in histopathology of resected schwannoma tumors16. Nearly all schwannoma cells display the classic long, spindled, bipolar cell shape associated with both Schwann and schwannoma cells6,17 however other cell morphologies, as described by Cravioto (Amoeboid Microglia-like Cells, Spindle-Shaped Cells, Racket-Shaped Cells, and Kite-Shaped Cells) sometimes occur14. Overall, cell growth and proliferation are very slow in our base medium (DMEM/10%FBS/Insulin/N2 supplement). Addition of known mitogenic factors such as forskolin and β1-neuregulin significantly increases cell proliferation18.
Critical Protocol Steps
Our schwannoma tissue culture protocol is simple and fairly forgiving; however there are several key steps that ensure success. First, correct tumor handling is critical. Expect best results when tumor is placed directly into ice-cold HBSS+/+ as soon as possible once removed from the patient. This is contrary to the usual surgical practice of collecting resected tumor in sterile saline then sending the aggregate tissue for processing in pathology at the end of the case. Second, keep tissue samples as cool as possible during processing. It is vital that the resected tumor is kept ice cold, and that processing occurs as expediently as possible following removal. Do as much of the tissue processing as possible on ice as well. Third, aggressively wash the tumor samples. This helps reduce the risk of bacterial or yeast contamination. Fourth, do not over-mince tumor specimens. Tumor trituration depends on a semi-subjective assessment of when to continue on with a smaller diameter P1000 pipette tip.
The authors have nothing to disclose.
Support: NIDCD R01DC009801, P30DC010362, 5T32DC000040-17
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 100mm 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 | |
15ml Conical Tube | Midwest Scientific | TP91015 | |
50ml Conical Tube | Midwest Scientific | TP91050 | |
2mL Round Bottom Tube | USA Scientific | 1620-2700 | |
Small Scissor | FST | 14058-11 | |
Small Forceps | FST | 11251-20 | |
Scalpel Handle | FST | 10004-13 | |
#11 Scalpel Blade | Roboz | RS-9801-11 | |
Non-Tissue Culture 100mm 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/out 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 (1mg/ml 200x) | Sigma | I6634 | Schwannoma Culture and Media Components |