B 27 electrophysiology

BS were generated as previously described^{18 (link)}. In order to incorporate glioblastoma cells into the BS, the protocol was slightly modified as follows: NPCs were grown (as above) in 175 mm² poly-l-ornithine and laminin-coated flasks. When NPCs were at 90% confluency, 7 × 10⁵ glioblastoma cells were plated on top of the NPCs. After 24 hours the cells were detached mechanically using a cell scraper (Sarstedt). The mixture of cells was pipetted repeatedly to disaggregate cell clumps. A density of 2 × 10⁶ cells per well were plated on a non-coated 6 plate-well. Cells were grown in differentiation medium (Neurobasal^® electro Medium (Gibco) supplemented with 5% B-27^® Electrophysiology (Gibco), 1% Glutamax (Gibco), 0.01 μg/ml human recombinant GDNF (Gemini), 0.01 μg/ml human recombinant BDNF (Gemini). Cultures were kept at 37 °C in an atmosphere of 5% CO₂ under constant gyratory shaking (88 rpm, 19 mm orbit) for up to 7 weeks, Fig. 1 Supplementary Data.

The CRL-2097 line was derived from CCD-1079Sk ATCC^® CRL-2097™ fibroblasts purchased from ATCC and was kindly provided by Dr. Hongjun Song within our joint NIH NCATS funded project (Pamies et al., 2017 (link); #1U18TR000547-01). The iPS2C1 line was kindly provided by Dr. Herbert Lachman. All studies followed Institutional Review Board protocols approved by the Johns Hopkins University School of Medicine. Differentiation from iPSCs to NPCs has been previously described (Wen et al., 2014 (link)). The BrainSpheres were generated as described in Pamies et al. (2017 (link)). Briefly, at 90% confluency, NPCs were detached mechanically and counted. The 2 × 10⁶ cells per well were plated in uncoated six well-plates. After 2 days, NPC medium was changed to differentiation medium (Neurobasal^® electro Medium (Gibco) supplemented with 2% B-27^® Electrophysiology (Gibco), 1% Glutamax (Gibco), 0.01 μg/ml human recombinant GDNF (Gemini), 0.01 μg/ml human recombinant BDNF (Gemini). Cultures were kept at 37°C in an atmosphere of 5% CO₂ under constant gyratory shaking (88 rpm, 19 mm orbit) for up to 8 weeks. The medium was partly exchanged three times a week.