Culturesure y 27632

The KhES-3 cell line was established at and provided by Kyoto University (Nakatsuji, 2005 (link)). The protocol of this study was reviewed by the Ethics Committee of CiRA in accordance with the "Guidelines for Derivation and Utilization of Human Embryonic Stem Cells" by the Ministry of Education, Culture, Sports, Science and Technology, Japan. The iPS cell lines were established from healthy Japanese donors at CiRA, Kyoto University, and were approved for use by the Ethics Committee of Kyoto University.
Since it has been reported that the toxicity of antioxidants such as catechin is suppressed in the presence of albumin (Zhang et al., 2016 (link)), maintenance culture was carried out for all cell lines including human ES cells using albumin-free Essential 8 Medium (Thermo Fisher Scientific) in six-well feeder-free culture dishes coated with 5 μg/mL vitronectin (VTN-N; Thermo Fisher Scientific). When seeding the cells, 10 μM CultureSure® Y-27632 (FUJIFILM WAKO) was added, and medium exchange on day 1 and thereafter was performed without Y-27632.

Human 1383D6 iPS cells (RIKEN BRC, #HPS1006) were electroporated with a combination of an AAVS1 donor vector (3 µg, KW1368) and an AAVS1 TALEN pair (1 µg each) (KW1295_pCAG-TALdNC-AAVS1-T1-pA, Addgene, #80495; KW1296_pCAG-TALdNC-AAVS1-T2-pA, Addgene, #80495)^{70 (link)}. Electroporation was performed using a NEPA21 electroporator (Nepa Gene Co. Ltd) with the following condition: Poring pulse (voltage: 125 V, pulse length: 5 ms, pulse interval: 50 ms, number of pulses: 2, decay rate: 10%, polarity: +); Transfer pulse (voltage: 20 V, pulse length: 50 ms, pulse interval: 50 ms, number of pulses: 5, decay rate: 40%, polarity: +/−). Subsequently, 5 × 10⁵ cells were seeded in a 6-cm dish in AK02N media containing CultureSure Y-27632 (Wako, #036-24023) and treated 48 h later with neomycin (G418 sulfate, 175 μg/mL, Merck, #345812) for eight consecutive days. Resistant cells were pooled and passaged into a single well of a 6-well plate. Single clones were isolated as Tet-ON dCas9 iPS cells (EP004-2-57 dCas9) and validated by genotyping PCR, Southern blotting, and mCherry expression upon Dox treatment (1 µg/mL).

To evaluate the influence of substrate stiffness on further proinflammatory responses of hGFs under inflammatory condition, LPS extracted from Escherichia coli O55:B5 (Sigma-Aldrich, St. Louis, MO, USA) was used. The cell cultures were added at a final concentration of 0, 10, 100, or 1000 ng/mL LPS at cell seeding or with medium change after 2 h of co-incubation with the following the cellular mechanotransduction inhibitor. The cells were co-incubated with LPS for 12 h at 37 °C in a 5% CO₂ atmosphere and then evaluated for proinflammatory responses.
To investigate the cellular signaling pathways involved in the induction of proinflammatory responses in gingival fibroblasts by substrate stiffness, two inhibitors with different points of action on cellular mechanotransduction were used. Y-27632 (CultureSure®Y-27632, FUJIFILM Wako Pure Chemical Corporation) and blebbistatin (B592500, Toronto Research Chemicals, Toronto, ON, Canada) inhibited ROCK and myosin II, respectively. After 10-h incubation, the hGF culture on the collagen-coated polystyrene culture plate or soft or hard PDMS was coincubated with 0, 5, or 10 μM Y-27632 or 0, 10, 30, or 50 μM blebbistatin for 2 h at 37 °C in a 5% CO₂ atmosphere. After 2-h of co-incubation with the inhibitor, hGF cultures were subjected to LPS co-incubation as described above.

The next day, cells were transferred from PMEA chamber slide to Matrigel-coated culture plates. Cells in the chamber slide were washed twice with PBS, and then overlaid with Gibco TrypLE^TM Express (Thermo Fisher Scientific), and incubated at 37 °C for 10 min. Cells were collected in standard medium supplemented with 10% FBS, and then centrifuged at 300× g for 5 min. After discarding the supernatant, cells were recovered with modified ES medium [48 (link)] and seeded on Matrigel (Corning, NY, USA)-coated 24- or 96-well plates. Fresh ES medium was added every 2 or 3 days.
The modified ES medium contained Advanced DMEM/F12 (Thermo Fisher Scientific), Penicillin-Streptomycin Solution Hybri-Max^TM (100 ng/mL; Sigma Aldrich, MO, USA), gentamycin sulfate solution (25 mg/mL, Wako Pure Chemical, Osaka, Japan), HEPES buffer solution (10 mM; Thermo Fisher Scientific), B-27 supplement (Thermo Fisher Scientific), bovine serum albumin (1 mg/mL; Sigma Aldrich), Glutamax (Thermo Fisher Scientific), recombinant murine Noggin (50 ng/mL; Pepro Tech, Cranbury, NJ, USA), recombinant human R-Spondin1 (500 ng/mL; R&D Systems, Minneapolis, MN, USA), recombinant murine EGF (50 ng/mL; Pepro Tech), recombinant mouse HGF protein (50 ng/mL; R&D Systems), recombinant mouse Wnt-3a protein (100 ng/mL; R&D Systems), Culture Sure Y-27632 (10 μM; Wako), and hydrocortisone (0.5 μM).