266 Chapter 10 3. Materials & Methods Ethics statement. The generation and characterisation of these hiPSC lines were approved by the Medical Ethical Committee (TCBio) of University Medical Center (UMC) Utrecht; approval number 12–387. The study was performed according to the approved ethics protocol, including the receipt of informed consent. PBMC collection and reprogramming. 9 ml of whole blood was collected from each donor into Lithium heparin Vacutainer blood collection tubes. PBMCs were isolated using Ficoll density gradient and cultured in StemPro-34 SFM with 100 ng/mL SCF, 100 ng/mL FLT-3, 20 ng/ mL TPO and 20 ng/mL IL-6 (Thermofisher Scientific). Reprogramming was performed using the Cytotune 2.0 Sendai Virus Kit (Thermofisher Scientific at the appropriate MOI (i.e., KOS MOI=5, hc-Myc MOI=5, and hKlf4 MOI=3). Colonies exhibiting hiPSC-like morphology were manually selected 2–3 weeks post-transduction and maintained on 0.1 mg/mL growth factor reduced Matrigel (Corning) and Essential 8 (Thermofisher Scientific) with a 7-day passage cycle in the first 1-5 passages, after that the splitting ratio was increased to 1:10-1:15. hiPSCs were cultured by a medium change every day and a 4-day passage cycle. To improve cell survival, split ratio reliability, and to reduce selective pressure, for the first 10 passages a concentration of 10 μM ROCK inhibitor (Calbiochem) was used. Thereafter the concentration of rock was decreased in steps till 20 μM was achieved. Maintenance of hiPSC. The hiPSCs were non-enzymatically passaged using 0.5 mM EDTA (Invitrogen, 15575-038) every 4 days. In brief, 1 ml 0.5 mM EDTA-PBS was added per 9.6 cm2 surface area. Cells were incubated at room temperature for 3-5 minutes until cells began to separate uniformly throughout the colonies. PBS-EDTA was removed and hiPSC colonies were washed off swiftly using 1 ml E8 medium. WhiPSC clumps were passaged in a splitting ratio of 1:10-1:15 routinely at 80% confluence. Additionally, 20 μM ROCK inhibitor (Calbiochem) was used in the first 24 hrs. To maximize the flexibility in experimental workflow, hiPSCs were cryopreserved and recovered as clumps or single cells using the PSC Cryopreservation Kit (Thermofisher Scientific). Immunocytochemistry. hiPSC were cultured on a Matrigel-coated 384 wells plate, fixed in paraformaldehyde (4%) for 15 minutes, and permeabilized in blocking/permeabilization buffer (5% BSA/0.3% Triton-X-100 in PBS) for 30 minutes. Primary antibodies were added as shown in Table 2 and incubated in 1:5 diluted blocking/permeabilization buffer DPBS overnight at 4°C. Cells were washed four times with PBS for 5 minutes and incubated with Alexa-conjugated secondary antibodies (life technologies) diluted in 1:5 diluted blocking/permeabilization buffer (Table 2) in the dark at room temperature for 1 hour. Cells were washed four times
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