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Non-colony type monolayer (NCM) culture of pluripotent stem cells

  • hESCs are initially grown on MEF feeder layer in routine hESC medium as colonies in 6-well plate (as detailed in the website of the NIH Stem Cell Unit).
  • 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 of MEF-conditioned medium (MEF-CM) (or mTeSR1 medium, from StemCell Technologies) 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 get rid of cell aggregates. Approximately 2 x 106 hESCs are seeded into one well (2.1 x 105 cells/cm2) of the 6-well plate coated with 2.5% hESC-qualified Matrigel (BD Bioscience) in MEF-CM supplemented with 100 ng/ml of FGF2 and 10 µM Y-27632 (or mTeSR1 medium with 10 µM Y-27632) for facilitating the initial 24-hour single-cell plating efficiency (SCPE).
  • Next day (within 24 hr), the medium is replaced with 50% regular hESC medium and 50% MEF-CM containing 4 ng/ml of FGF2 (or 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 few days with medium change daily. At day 4 or 5 (depending on the confluence of the culture for the initial adaptation process), the cells are again dissociated in 1 ml Accutase for the next passage (plated at a cell density of 1.35to 2.1 x 105 cells/cm2) or prepared for desired experiments.
  • The schedule for passaging the adapted hESCs with the NCM culture may 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 cells reach confluence and cell borders disappear. A 1 to 3 splitting ratio (i.e., 1 confluent well of cells for plating on 3 wells) is recommended.
  • Cell freezing: a confluent well (~ 5 to 6 x106 cells) is used for one frozen vial: The cells are dissociated with 1 ml Accutase, diluted with 5 ml medium, and centrifuged as above. The pellet is resuspended in 500 µl of hESC-CM and mixed with 500 µl of 2X hESC freezing medium (i.e., 60% FBS, 20% DMSO, and 20 µM Y-27632 in hESC-CM or mTeSR1) in a cryopreservation vial.
  • Cell thawing: cells in one cryopreservation vial are used for plating on 1 well in 6-well plate. The cells are thawed in a 37°C water bath for 2 min, transferred to a 15 ml tube containing 5 ml pre-warmed hESC-CM (or mTeSR1) and 10 µM Y-27632, and centrifuged as described above. Cell pellets are resuspended with hESC-CM (or mTeSR1) containing 10 µM Y-27632 and plated. Medium is changed daily and cells will be confluent 1-3 days after plating.


Chen KG, Mallon BS, Hamilton RS, Kozhich OA, Park K, Hoeppner DJ, Robey PG, McKay RD. Non-colony type monolayer culture of human embryonic stem cells. Stem Cell Res. 2012 Jun 28; 9(3):237-248. [Epub ahead of print]