Ascorbic acid a4403

The work is approved by Institutional Review Boards (IRB) at Mackay Memorial Hospital (MMH), IRB approval number: 14MMHIS261, and the Institutional Animal Care and Use Committee (IACUC) of National Taiwan University (NTU), IACUC approval number: NTU-103-EL-92. All the participants in this study understand the research and the written consent forms have been obtained from the patients. Patients were subjected to the controlled ovarian hyperstimulation (COH) treatments at MMH using the GnRH antagonist protocol as described [19 (link)]. Clinically discarded CCs were collected with patients’ consent. Basic information of the patients is shown in Table 1. Freshly collected CCs from the same patient were pooled and immediately plated on matrigel (354234, Corning, NY, USA) coated 96-well dish in culture medium containing 2% fetal bovine serum (FBS, 16000–044), 0.05 mM ascorbic acid (A4403, Sigma-Aldrich, St. Louis, MO, USA), 0.05 μM dexamethasone (D4902, Sigma), 20 ng/mL epidermal growth factor (EGF, E9644, Sigma), basic fibroblast growth factor (bFGF, 13256–029, 50 ng/mL) and 1U/mL follicle stimulating hormone (FSH, F2293, Sigma). Medium was changed every 2–3 days after the cells attached on the dish. Twenty days post seeding, confluent cells were trypsinized by 0.05% Trypsin-EDTA. The cultured CCs at Passage 2 were subjected for hc-iPSC derivation.

Medium and supplements for cell culture were purchased from Biochrom AG (Berlin, Germany) or GIBCO Invitrogen (Karlsruhe, Germany). All reagents (e.g., Ascorbic acid A4403; Trypan Blue T8154), except those specified below, were purchased from Sigma-Aldrich (Deisenhofen, Germany). Capsazepine was purchased from Cayman Chemical Company (Ann Arbor, Mi, USA). The internal and external solutions for planar patch-clamping were provided by Nanion Company (Munich, Germany). Fura2/AM was purchased from PromoCell (Heidelberg, Germany).