Dmem ham s f 12

The TkDN4-M (4M) hiPSCs line was a kind gift from Dr. M Ohtsu at The Institute of Medical Science, The University of Tokyo. 4M cells were cultured as previously described with minor modifications [23 (link)]. They were maintained on mitomycin C (MMC; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan)-treated SNL feeder cells in hiPSCs medium (DMEM/Ham`s F12 (FUJIFILM Wako) supplemented with 20% Knockout Serum Replacement (KSR; GIBCO BRL, Palo Alto, CA, USA), 1x MEM nonessential amino acids (FUJIFILM Wako), 0.5x penicillin streptomycin(PS; FUJIFILM Wako), 55μM 2-melcaptoethanol (2ME Gibco) and 7.5μg/ml basic fibroblast growth factor (FGF2; Peprotech, Rocky Hill, NJ, USA). For passage, 4M colonies were detached using CTK solution, chipped by pipetting, and seeded onto MMC-treated SNL feeder (ECACC, Salisbury, UK) in hiPSCs medium once a week. The 15M63 hiPSCs line was kindly provided from the Center for iPS Cell Research and Application of Kyoto University (Kyoto, Japan) and maintained on vitronectin (Invitrogen, CA, USA) coated dishes in StemFit. For passage, 15M63 was dissociated with accutase (Innovative Cell Technologies, San Diego, USA) by pipetting and seeded on vitronectin coated dishes in StemFit (Ajinomoto, Tokyo, Japan) supplemented with 10μM Y27632 (Cayman Chemical, Ann Arbor, MI, USA).

The SH‐SY5Y human neuroblastoma cell line (ATCC^® CRL‐2266^TM) was purchased from the American Type Culture Collection. Cell culture and differentiation were performed as described previously (Tagai et al., 2020 (link)). In brief, SH‐SY5Y cells were cultured in Dulbecco's modified eagle medium (D‐MEM)/Ham's F‐12 (FUJIFILM Wako Pure Chemical Corporation, Cat#048‐29785) containing 10% fetal bovine serum (FBS), 100‐units/ml penicillin, and 100‐μg/ml streptomycin in a 37°C incubator in an atmosphere of 5% CO₂ and 100% relative humidity. In the cell differentiation, SH‐SY5Y cells were inoculated at an initial density of 1.0 × 10⁵ cells/dish in a collagen I‐coated φ 3.5 cm dishes. All‐trans‐RA (FUJIFILM Wako) was added 2 days after plating at a final concentration of 10 μM dissolved in high‐glucose D‐MEM (FUJIFILM Wako, Cat#043‐30085), which was supplemented with 15% FBS. After 5 days of culturing in the presence of RA, cells were washed with high‐glucose D‐MEM and incubated for 2 days with 100 ng/ml human recombinant BDNF (FUJIFILM Wako) in high‐glucose D‐MEM without L‐glutamine and phenol red (FUJIFILM Wako, Cat#040‐30095) containing 4 mM sodium pyruvate and 1 mM L‐glutamine. We prepared ndSH‐SY5Y cells using a modified differentiation protocol according to previously described methods (Agholme et al., 2010 (link); Encinas et al., 2000 (link); Krishtal et al., 2017 (link)).

Incisor enamel organ epithelial cells were isolated and enzymatically processed in 4w/v% collagenase (Fujifilm Wako Chemicals) in Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F12 (Fujifilm Wako Chemicals) for 30 min at room temperature, 0.5w/v% trypsin–EDTA (Fujifilm Wako Chemical) for 10 min at 37 ℃ followed by neutralization with DMEM/Ham’s F12 containing 10% fetal bovine serum (FBS). Single cell suspensions were filtered with a cell strainer (40 μm, Corning), and then subjected to fluorescence-activated cell sorting (FACS) to isolate tdTomato positive and negative cells using FACSMelody (BD Biosciences). Sorted cells were centrifuged 1000 rpm for 5 min and pellets were lysed in RA1 buffer (Macherey–Nagel) for RNA extraction.

EBV-negative HSC1 and SCC25 cells, as well as EBV-positive HSC1-EBV and SCC25-EBV cells [6 (link)], were cultured in Dulbecco’s Modified Eagle Medium (DMEM)/Ham’s F-12 (Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS) (HyClone^TM, GE Healthcare Life Sciences, Logan, UT, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Nacalai Tesque Inc., Kyoto, Japan). The cells were cultured at 37 °C in a humidified incubator with 5% CO₂.

SH-SY5Y cells are often employed as the in vitro models for anxiety [40 (link)] and pain [33 (link),41 (link)]. The SH-SY5Y cell lines (ATCC, Manassas, VA, USA) were maintained in DMEM/Ham’s F12 (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 2.5 mM L-glutamine, 10% fetal bovine serum, and antibiotics—100 U/mL of penicillin and 100 μg/mL of streptomycin (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan)—in a humidified atmosphere of 5% CO₂ at 37 °C. For the neuronal induction of SH-SY5Y cell lines, the culture medium was replaced with a differentiation medium containing 10 μM of all-trans retinoic acid (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) in the presence of Rice-koji extracts (10 or 500 µg in 1 mL PBS), EGT (10 or 100 µg in 1 mL PBS), or PBS alone. The differentiation medium was replaced every 5 days over 4 weeks of incubation [42 (link)].