Cytotune ips sendai reprogramming kit

Cord blood mononuclear cells (CBMCs) were directly obtained from the Cord Blood Bank of Seoul St. Mary’s Hospital. The Institutional Review Board (IRB) of the Catholic University of Korea, Seoul St. Mary’s Hospital approved this study. The reprogramming of CBMCs into iPSCs was induced using the Cytotune-iPS Sendai Reprogramming Kit (Life Technologies, Carlsbad, CA, USA). Briefly, CBMCs were seeded in a 24-well plate (3 × 10⁵ cells/well) with StemSpan™ medium (STEMCELL Technologies, Seattle, WA, USA). After addition of the viral components, the plate was centrifuged at 1160×g at 25 °C for 30 min and then incubated at 37 °C in 5% CO₂. On the next day, the cells were transferred to a 12-well plate coated with vitronectin (Life Technologies). The plate was centrifuged at 1160×g at 25 °C for 10 min, and Essential 8™ medium (Thermo Fisher Scientific, Waltham, MA, USA) was added at a 1:1 ratio. The cells were maintained in E8 medium until iPSC colonies were generated. The colonies were maintained in E8 medium (Thermo Fisher Scientific) on vitronectin-coated culture dishes. iPSCs were passaged every 3–4 days using Accutase Cell Detachment Solution (Global Cell Solutions, North Garden, VA) with Y-27632 dihydrochloride (R&D Systems, Minneapolis, MN, 10 µM). The medium was changed every day.

PBMCs were counted (3 × 10⁵ per well) and suspended in fresh StemSpan medium. Sendai viral particle factors from CytoTune-iPS Sendai Reprogramming Kit (A16518, Life Technologies, Carlsbad, CA, USA) were added based on the manufacturer's recommendations (3 × 10⁵ cell infectious units (CIU) of each particle per well, multiplicity of infection (MOI) = 7.5) [12 (link)]. Cells were centrifuged at 1,160 ×g, at 35°C, for 30 minutes. The cells were then incubated overnight in 5% CO₂ at 37°C. The next day, cells were transferred onto a vitronectin (A14700, Life Technologies) coated 12-well plate and settled by centrifugation for 10 minutes at 1,160 ×g. After centrifugation, TeSR-E8 medium (5940, STEMCELL Technologies) was added. To maintain reprogrammed cells, TeSR-E8 medium was changed daily in vitronectin-coated dishes.

To generate iPSCs, PBMCs and skin cells were obtained from RA patients with hepatotoxicity caused by MTX treatment, as well as from healthy control subjects. iPSCs were generated as previously described [24 (link)]. Briefly, PBMCs obtained from each group were cultured for 4 days in StemSpan medium (STEMCELL Technologies, Vancouver, Canada) to expand CD34-positive cells. Expanded PBMCs or skin cells were transfected with the CytoTune-iPS Sendai Reprogramming Kit (Life Technologies, Carlsbad, CA, USA) including the Yamanaka factors (Oct4, Sox2, KLF4, and c-Myc). PBMCs were induced to form iPSCs by centrifugation; the resultant attached cells were expanded and purified by colony picking.