(Basic protocol using the mTeSR1 medium)
- HESCs are initially grown on MEF feeder layers in routine hESC medium as colonies in 6-well plates (as detailed on the NIH Stem Cell Unit website).
- Colonies are dissociated by collagenase IV for 15 to 30 min.
- HESC pellets are washed once in D-PBS and then incubated with 1 ml 1X Accutase (Innovative Cell Technologies, Inc., San Diego, CA) for 15 min.
- The enzymatic reactions are briefly terminated by resuspending the cells in 5 ml mTeSR™1 medium (StemCell Technologies Inc. Vancouver, Canada) and centrifuged at 220 x g for 5 min.
- The dissociated single cells are filtered through a 40-µm BD Falcon™ Cell Strainer (BD Bioscience) to remove cell aggregates. Approximately 1.3 to 2 x 106 hESCs are seeded into one well (1.4 to 2.1 x 105 cells/cm2) of a 6-well plate coated with 2.5% hESC-qualified Matrigel (BD Bioscience) in mTeSR1 medium in the presence 10 µM Y-27632 (EMD Millipore) for facilitating the initial 24-hr single-cell plating efficiency (SCPE).
- Next day (within 24 hr), the medium should be replaced with mTeSR1 medium. Approximately, 40 to 85% of SCPE may be achieved at this stage.
- The cells are allowed to grow as a single-cell-formed monolayer for a few days with daily medium change. At day 4 or day 5 (depending on the confluence of the culture for the initial adaptation process), the cells will be re-dissociated in 1 ml Accutase™ (StemCell Technologies Inc.) for the next passage or prepared for desired experiments.
- The day schedule for passaging the adapted hESCs with NCM could be varied depending on cell density. We prefer to passage the cells at day 3 or 4 in the presence of 10 µM Y-27632, when cell growth reaches confluence and cell-to-cell borders disappear. A 1 to 3 splitting ratio (i.e., 1 confluent well of cells replated in 3 wells) would be fine.
- Cell freezing: a confluent well (~ 5 to 6 x106 cells) is used for one frozen vial: The cells are dissociated with 1 ml Accutase, resuspended in 5 ml medium, and centrifuged as above. The pellet is resuspended in 500 ul of mTeSR medium and mixed with 500 ul of 2X hESC freezing medium (i.e., 60% FBS, 20% DMSO, and 20 µM Y-27632 in mTeSR1 medium) in a cryopreservation vial. Alternatively, the cells may be frozen in CryoStor™ medium (StemCell Technologies Inc.) in the presence of 10 µM Y-27632. Please follow the vendor's instruction.
- Cell thawing: the cells in one cryopreservation vial are used for plating on 1 well of a 6-well plate. The cells are thawed at a 37°C water bath for 2 min, transferred to a 15 ml tube containing 5 ml pre-warmed mTeSR1 medium containing 10 µM Y-27632, and centrifuged as described above. The pellets are gently resuspended with mTeSR1 containing 10 µM Y-27632 (note: please do not produce air bubbles). The medium is changed daily. The cells would be expected to reach confluence on day 3 or 4.
Chen KG, et al. Non-colony type monolayer culture of human embryonic stem cells. Stem Cell Res. 9(3):237–48; November 2012.