Dmem medium

Human fetal bladder tissue derived CCC-HB-2 cell line, human bladder epithelial immortalized cell line SV-HUC-1 and BC cell lines (T24, J82, UMUC3) were stored in our laboratory and identified by STR Authentication. T24/gem and J82/gem cell lines (resistant to gemcitabine) were established in previous studies 20 (link),21 (link). All cells were cultured in DMEM medium with 10% fetal bovine serum (ExCell Bio, Shanghai, USA) in a 37°C incubator.

Isolated brainstems were prepared from neonatal (P0-P1) SD rats using previously described methods (Mazza et al., 2000 (link)). A microscope was used for tissue dissection under aseptic conditions. The rat pups were decapitated, and the brainstems were rapidly removed. After microdissection of the cerebellum and pons from the brainstem, the medulla oblongata was separated and transferred to a new chamber. The tissue containing the medulla oblongata was minced and digested enzymatically with 0.25% trypsin for 15 min. The medulla oblongata cells were filtered, then dispersed and seeded in DMEM medium supplemented with 10% fetal calf serum and 10% equine serum (ExCell Bio, Shanghai, China). Twenty-four hours after seeding, the medium was replaced by Neurobasal (Gibco, Shanghai, China) Medium with B27 (Gibco, Shanghai, China) supplement (50:1). The medulla oblongata cells were fed on days 3–7 with a half volume change of Neurobasal/B27 medium containing Ara-C (3–5 μM) to inhibit glial growth. The cells were cultured for 10–15 days prior to use.