Protocol for Transitioning to mTeSR™ Plus
- Aug 2019
The following protocol is for transitioning human ES or iPS cells from feeder-free media (e.g. mTeSR™1) to mTeSR™ Plus (Catalog #05825). For complete instructions, refer to the Technical Manual: Maintenance of Human Pluripotent Stem Cells in mTeSR™ Plus (Document #23032) or contact us to request a copy.
This protocol references specific sections in the Technical Manual: Maintenance of Human Pluripotent Stem Cells in mTeSR™ Plus (Document #23032).
- Coat a 6-well tissue culture plate with Corning® Matrigel® (section 4.2.1) or Vitronectin XF™ (section 4.2.2). Add 2 mL medium/well to the coated plate as indicated below:
- Generate a cell aggregate solution using either ReLeSR™ (section 5.1) or Gentle Cell Dissociation Reagent (section 5.2).
- Plate the cell aggregate mixture as follows:
- Place the plate in a 37°C incubator. Move the plate in several quick, short, back-and-forth and side-to side motions to distribute the cell aggregates. Do not disturb the plate for 24 hours.
- Perform medium changes as indicated below. Visually assess cultures every 1 - 2 days to monitor growth.
- Based on the visual assessment (section 3.2), determine when the cultures are ready to passage. The appropriate passaging day for the mTeSR™ Plus cultures may be earlier than with mTeSR™1 cultures.
- At the time of passaging, select the mTeSR™ Plus well that is ready to be split; maintain the split ratio of the selected well. If overgrowth is observed, decrease plating density by 20%.
NOTE: The dissociation incubation times may need to be adjusted; dissociation with non-enzymatic passaging reagents should be monitored under the microscope until the optimal time is determined.