Primary culture of human Schwann and schwannoma cells: Improved and simplified protocol

Abstract

Primary culture of human Schwann cells (SCs) and vestibular schwannoma (VS) cells are invaluable tools to investigate SC physiology and VS pathobiology, and to devise effective pharmacotherapies against VS, which are sorely needed. However, existing culture protocols, in aiming to create robust, pure cultures, employ methods that can lead to loss of biological characteristics of the original cells, potentially resulting in misleading biological findings. We have developed a minimally manipulative method to culture primary human SC and VS cells, without the use of selective mitogens, toxins, or time-consuming and potentially transformative laboratory techniques. Schwann cell purity was quantified longitudinally using S100 staining in SC cultures derived from the great auricular nerve and VS cultures followed for 7 and 12 weeks, respectively. SC cultures retained approximately ≥85% purity for 2 weeks. VS cultures retained approximately ≥80% purity for the majority of the span of 12 weeks, with maximal purity of 87% at 2 weeks. The VS cultures showed high level of biological similarity (68% on average) to their respective parent tumors, as assessed using a protein array featuring 41 growth factors and receptors. Apoptosis rate in vitro negatively correlated with tumor volume. Our results, obtained using a faster, simplified culturing method than previously utilized, indicate that highly pure, primary human SC and VS cultures can be established with minimal manipulation, reaching maximal purity at 2 weeks of culture. The VS cultures recapitulate the parent tumors' biology to a great degree, making them relevant models to investigate VS pathobiology.

Nonsteroidal anti-inflammatory medications are cytostatic against human vestibular schwannomas

“…Details of the simplified culture method are provided in (17). Briefly, for SC cultures, GAN samples were washed with sterile PBS thrice to remove accompanying blood or scar tissue, and transferred to supplemented DMEM/F12 medium, consisting of 44% Dulbecco's Modified Eagle's Medium (DMEM; Life Technologies, Grand Island, NY), 44% F12 Nutrient Mixture (ThermoScientific, Waltham, MA), 10% Fetal Bovine Serum (Life Technologies, Grand Island, NY), 1% Penicillin/Streptomycin mix (ThermoScientific, Waltham, MA, #15140-122) and 1% GlutaMAX (Life Technologies, Grand Island, NY).…”

“…The same protocol was followed for VS cell cultures with the only major difference being an 18 h enzymatic incubation rather than the 24 h used here for healthy SCs (17). …”

“…Proliferation was assessed in cultured cells as described in (17). Briefly, BrdU was added to the cells at a concentration of 10 μg/ml 20 hours before the cells were fixed.…”

Interplay between VEGF-A and cMET signaling in human vestibular schwannomas and schwann cells

“…15 Briefly, using sterile technique under the hood, freshly harvested VS or GAN tissue was rinsed in sterile PBS trice and cut into 1 mm-sized pieces in Dulbecco's modified eagle's medium with Ham's F12 nutrient mixture (DMEM/ F12), 10% fetal bovine serum, 1% Penicillin/Streptomycin (Pen/Strep) and 1% GlutaMAX (all purchased from Life Technologies). To obtain a more pure SC population, the epineurium was removed from the nerve tissue by tugging and removing the outer layers under a dissecting microscope.…”

A novel alternative splicing isoform of NF2 identified in human Schwann cells

“…The first studies that used primary cultured NF2-deficient schwannoma cells isolated from human tumors found unique mutant phenotypic traits at the cellular level, such as cytoskeletal abnormalities (21) and growth characteristics (22). However, these cells grow extremely slowly and it is challenging to transfect them with DNA, therefore rendering biochemical and mechanistic studies difficult (23). By contrast, the HEI-193 cell line does not have these disadvantages.…”