Transitioning Pluripotent Stem Cells from Feeder-Free Media to mTeSR™ Plus
The following protocol is for transitioning human embryonic stem (ES) and induced pluripotent stem (iPS) cells from feeder-free media (e.g. mTeSR™1) to mTeSR™ Plus, a serum-free, stabilized cell culture medium. mTeSR™ Plus allows for greater cell expansion rates with daily feeding, while also maintaining cell quality during restricted feeding schedules. For complete instructions, refer to the Technical Manual: Maintenance of Human Pluripotent Stem Cells in mTeSR™ Plus or contact us to request a copy.
- Cell Culture Matrix (e.g. Vitronectin XF™, Catalog #07180, or Corning® Matrigel®)
- 6-well Tissue Culture Plate. The type of plate required will depend on your desired matrix:
- Dissociation Reagent (e.g. ReLeSR™, Catalog #05872, or Gentle Cell Dissociation Reagent Catalog #07174)
- mTeSR™ Plus (Catalog #05825)
- mTeSR™1 (Catalog #85850)
Before You Begin: This protocol references specific sections in the Technical Manual: Maintenance of Human Pluripotent Stem Cells in mTeSR™ Plus.
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:
- For wells in column 1: Use current mTeSR™1 plating density/split ratio
- For wells in column 2: Use current mTeSR™1 plating density/split ratio
- For wells in column 3: Decrease current plating density by ~25%
- 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.
- mTeSR™1: Perform daily medium changes.
- mTeSR™ Plus: Medium can be changed daily or every other day. To skip two consecutive days of feeding, add twice the volume of medium.
- 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.