Culturing Human Cell Lines for Research

Normal human astrocytes (NHA) cell line was purchased from Gibco (N7805100) and cultured in human astrocytes growth medium (Cell Applications). Human neuroblastoma (SH-SY5Y) cell line was obtained from ATCC (CRL-2266) and cultured in DMEM/F12 medium (Gibco). Human glioblastoma (U118) cell line was purchased from ATCC (HTB-15) maintained in Dulbecco's Modified Eagle's Medium (Gibco). In addition, human neural progenitor cells (hNPCs; ReNcell VM) (Takahashi and Yamanaka 2006 (link); Donato et al. 2007 (link)) were commercially purchased from Sigma-Aldrich (SCC008). The cells were grown on 20 μg/mL laminin (Merck)-coated culture plates containing ReNCell NSC maintenance medium (Merck) supplemented with 20 ng/mL of bFGF and EGF (Merck). All culture media were supplemented with 10% fetal bovine serum (FBS) and 1× antibiotic-antimycotic (Gibco) at 37°C with 5% CO₂. Cells were passaged when the confluence reached 80% of the culture plate every 3 d. Briefly, cells were rinsed with PBS and then incubated in Accutase (Millipore) for 3–5 min until the cell detached. We used the culture medium to inhibit enzymatic reaction and centrifuged the suspension at 500g for 5 min. We then resuspended the cell pellet in fresh medium and counted the cell number.