Establishment and phenotyping of neurosphere cultures from primary neuroblastoma samples

Background: Primary cell culture using serum free media supplemented with growth factors has been used in a number of cancers to propagate primary cells with stem like properties, which form as spherical cellular aggregates. Methods: We systematically evaluated the capacity of freshly disaggregated neuroblastoma tumors to become established as neurospheres in stem cell media using a uniform protocol. 67 primary neuroblastoma samples from patients treated at a single institution were prospectively evaluated for their ability to become established in culture. Samples, either solid tissue or cells from surgical transit fluid both post chemotherapy and chemotherapy naïve, were evaluated from diagnostic needle biopsies or surgical resections. Results: Overall 37 neurosphere cultures were successfully established from 67 samples. In 11 out of 14 cases investigated by flow cytometry, uniform staining for neuroblastoma markers CD56 and GD2 was demonstrated in CD45 negative non-hemopoietic cells, confirming neuroblastoma origin. Conclusion: We present a simple and reproducible approach for producing primary neurospheres from neuroblastoma samples, which provides a reliable resource for future work including genetic analysis, stem cell research and models for therapeutics.