Cryostor cs10 freezing media

For the static culture, the eCB-MSCs were expanded in 75cm² T-flasks (Falcon Cat#: 353136) at an inoculation density of 5000 cells/cm², with 12 mL of media, in a humidified incubator (37 °C and 5% CO₂ in ambient air). Once the cells neared confluence (~ 80%), they were harvested by exposing the cells to 0.25% Trypsin for 5 min in a humidified incubator (37 °C and 5% CO₂ in ambient air), followed by deactivation of the Trypsin using FBS containing media. The cells were then enumerated on a haemocytometer using 0.1% Trypan Blue exclusion, and either passaged onto new T-flasks, inoculated on microcarrier beads within bioreactors, or cryopreserved in Cryostor CS10 freezing media (BioLife Solutions Cat # 210102) for future cell characterization.

At day 1, a 90% confluent flask of hPSCs was dissociated with StemPro^® Accutase^® (Gibco #A1110501), and inoculated in 125 mL Erlenmeyer flasks (Corning #431143) containing 20 mL of E8 medium (in house) with 10 µM Y-27632 (Tocris #1254) at a cell density of 3.33 × 10⁵ cells/mL for hiPSCs and 6.67 × 10⁵ cells/mL for hESCs. Flasks were placed in an incubator on an orbital shaker (Celltron, Infors HT) rotating at 70 rotations per minute (rpm). Medium was changed every 24 h (h). For each medium change, the aggregates were collected and centrifuged (Multifuge 3SR+, Thermo) at 300 g for 2 min before placing them in fresh medium. The following day (day 0), the media was changed to RPMI 1640 (Gibco #21875034), 100 ng/mL activin A (PeproTech #120-14E), and 3 µM CHIR99021 (provided by the Institute of Organic Chemistry, Leibniz University, Hannover, Germany). On day 1, the medium was changed to 100 ng/mL activin A, and 0.8% Knockout™ Serum Replacement (KSR; Gibco #10828028) in RPMI 1640. On day 2, the medium was changed to 100 ng/mL activin A, 8% KSR in RPMI 1640. On day 3 of differentiation, aggregates were dissociated with Accutase for 3 min in a water bath at 37 °C, analyzed for DE markers, and further differentiated into liver, pancreatic, intestinal, and lung progenitor cells. Dissociated day 3 cells were also frozen in CryoStor^® CS10 Freezing Media (BioLife Solutions #210102).

Six days before the start of differentiation, pluripotent stem cells were passaged in flasks at a low density, 1.2 × 10⁴ cells/cm², using E8 medium and 10 µM Y-27632 (Tocris #1254). Medium was changed every 24 h. At day 3, medium was changed to E8 medium and Supplement 1, supplied in the SD kit (STEMCELL Tech. #05115). On day 1 of differentiation, cells were dissociated with StemPro^® Accutase^® (Gibco #A1110501) and inoculated in shaker flasks, at a cell density of 3.33 × 10⁵ cells/mL for hiPSCs and 6.67 × 10⁵ cells/mL for hESCs, in E8 medium, E8 supplement (STEMCELL Tech. #05116), and 10 µM Y-27632. For each medium change, the aggregates were collected and centrifuged (Multifuge 3SR+, Thermo) at 300× g for 2 min before placing them in fresh medium. On day 0, medium was changed to STEMDiff™ Endoderm Basal Media containing Supplement MR and CJ. On day 1 and day 2, aggregates were fed with STEMDiff™ Endoderm Basal Media containing Supplement CJ only. On day 3, aggregates were dissociated and analyzed for DE markers, and also further differentiated into liver, pancreatic, intestinal, and lung progenitor cells. Dissociated cells were also frozen in CryoStor^® CS10 Freezing Media (BioLife Solutions #210102) at 6 × 10⁶ cells/vial.