Cellstart cts substrate

Human umbilical cords (hUCs) were collected from West China Women’s and Children’s Hospital without any complications of pregnancy or parturition, and the collected hUCs were transferred in sterile boxes that contained cold Hanks’ Balanced Salt Solution (HBSS) (Life Technologies, USA). Written informed consent was obtained from the pregnant women before labor. This study was approved by the institutional ethics committee of Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College (CAMS & PUMC).
hUCs were dissected longitudinally, and the arteries and veins were removed. The remaining pieces were chopped into 0.2cm³ size. These explants were transferred in the CELLstart™ CTS™ Substrate (Life Technologies, USA)-coated 100 mm plates (Nunc, Denmark). Then, a few StemPro® MSC SFM XenoFree medium (Life Technologies, USA) with 1% penicillin-streptomycin (Life Technologies, USA) was added to the plates, and the explants were cultured at 37°C in a 5% CO₂ incubator and left undisturbed to allow the cells to migrate from the explants. After 7–9 days, the MSC-like cells were found around the fragments. The cells were passaged into another plates and further split 1:4 by 0.05% Trypsin-EDTA (Life Technologies, USA) once the cells reached 80% confluence.

The use of the human umbilical cord tissue from a healthy donor, written informed consent was obtained from the pregnant women before labor. The approval of human umbilical cord-derived MSCs was obtained by the institutional ethics review committee of Institute of Blood Transfusion, Chinese Academy of Medical Sciences, China (approval number: 202030). The human UC-MSCs were manufactured as previously described [23 (link)]. Human umbilical cords were dissected longitudinally, and the arteries and veins were removed. The remaining pieces were chopped into 0.2 cm³ sections [23 (link)]. These explants were transferred in the CELLstart™ CTS™ Substrate- (Life Technologies, USA) coated 100 mm plates (Nunc, Denmark). StemPro1 MSC SFM XenoFree medium (Life Technologies, USA) with 1% penicillin streptomycin (Life Technologies, USA) was added to the plates, and the explants were cultured at 37 °C in a 5% CO2 incubator and left undisturbed to allow the cells to migrate from the explants. After 7–9 days, MSC-like cells were found around the fragments. The cells were passaged into another plate and further split 1:4 using 0.05% Trypsin–EDTA (Life Technologies, USA) once the cells reached 80% confluency.

CTS CELLstart substrate (A1014201), CytoTune-iPS 2.0 Sendai reprogramming kit (A16517), Hoechst 33342 trihydrochloride (H3570), LysoTracker red DND-99 (L7528), PSC neural induction medium (A1647801), Nile red (N-1142), StemPro NSC SFM (A1050901), and StemFlex medium (A3349401) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). ROCK inhibitor Y-27632 (1284) was purchased from Tocris Bioscience (Ellisville, MO, USA). Matrigel (354277) was obtained from Corning (Corning, New York, NY, USA). Accutase (07920), and mTeSR1 medium (85850) were purchased from StemCell Technologies (Vancouver, BC, Canada). Delta-tocopherol was purchased from Sigma-Aldrich (St. Louis, MO, USA) and purified by HPLC to a purity greater than 99% [20 (link)]. Dermal fibroblast lines (GM20122, GM00248, and GM00338) were obtained from the NIGMS Human Genetic Cell Repository at the Coriell Institute for Medical Research.