Figure 1. Subpopulations of murine N2a and human CHLA-02-ATRT cells form multicellular tumour spheroids during surface-associated culture.

(A) Representative image of an N2a MCTS. N2a cells were plated at a density of 1×10³ cells per 60 mm dish and cultured under standard conditions for 5 days. Tumour spheroids (S) were differentiated from monolayer cells (M) at 6 days and exhibited a rough 3-dimensional appearance comparable to MCTS structures grown on non-adherent surfaces or in suspension. Scale bar = 100 µm. (B) MCTS production increased with serial passage. N2a and CHLA-02-ATRT cells were plated at a density of 1×10³ cells per 60 mm dish and cultured for 6 days. MCTS number was recorded. Cells were scraped from the dish surface, treated with non-enzymatic cell dissociation solution and strained over a 70 µm filter to produce a single-celled suspension which was verified with a haemocytometer. New 60 mm dishes were plated with 1×10³ cells from this suspension and cultured for a further period of 6 days. MCTS number was again recorded. This procedure was repeated for a total of 7 passages. Statistical differences were calculated by Student's t-test. Each bar represents the mean ± s.e.m. for at least three independent experiments. Significant differences (P<0.001) are indicated by asterisks (*).