Luhmes

Human embryonic kidney cells (HEK) cell line was purchased from ATCC (ATTCC CRL-1573 VA, USA). The cells were maintained in Dulbecco’s minimum essential medium (11955-065 Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (F2442, Sigma Aldrich, St. Louis, MO, USA) and 1% penicillin/streptomycin (15149-122, Life Technologies, Carlsbad, CA, USA). Lund human mesencephalic (LUHMES) cells (ATCC CRL 2927 VA, USA) were gifted from Dr. David Bloom’s lab and cultured as per Edwards and Bloom, 2019 [18 (link)]. Briefly, cells were cultured in proliferation medium with FGF (100-18B Peprotech, NJ USA) supplement for 72 h while changing the medium after every 24 h. Then, tetracycline (Sigma Aldrich, St. Louis, MO, USA) containing a differentiation medium was added and changed every 24 and 48 h for 4 days post incubation to induce the neuronal cell differentiation. Formation of neurite threads was confirmed under the microscope and through Western blot analysis with marker antibody (βIII-Tubulin TU-20 (4466S Cell Signaling Technology, Danvers, MA, USA) expression.

LUHMES (ATCC^® CRL_2927™) 3D cell culture and differentiation protocol were followed as described (Harris et al. 2017 (link)). Briefly, cells were used between passages 15 and 25. 4 × 10⁶ cells were placed in a 175 cm² flask for 48 h to expand cells. On day 0, 3D-differentiation was initiated: 5.5 × 10⁵ cells were seeded into each well of a 6-well plate and placed on a gyratory shaker at 80 rpm (50 mm orbit diameter) in an incubator at 37 °C, 10% CO₂, and 95% humidity.