Successful culture of human embryonic stem (ES) and induced pluripotent stem (iPS) cells requires the use of a suitable matrix to allow attachment of cell aggregates. For example, Vitronectin XF™ provides a defined culture system, allowing complete control over the culture environment. This results in more consistent cell populations and reproducible results in downstream applications. This protocol is for coating cultureware with Vitronectin XF™, which can be used for ES and iPS cells cultured in mTeSR™1, mTeSR™ Plus, or TeSR™-E8™. For coating cultureware with other matrices, refer to section 4.2.1 (Corning® Matrigel®) or 4.2.3 (CellAdhere™ Laminin-521) of the Technical Manual: Maintenance of Human Pluripotent Stem Cells in mTeSR™ 1.

Materials

• Vitronectin XF™ (Catalog #07180)
• CellAdhere™ Dilution Buffer (Catalog #07183)
• 50 mL polypropylene conical tube (e.g. Falcon® Conical Tubes, 50 mL, Catalog #38010)
• Non-tissue culture-treated cultureware (e.g. Non Tissue Culture-Treated 6-Well Plates, Catalog #27147)
• Cell culture medium (e.g. mTeSR™1, Catalog #85850, mTeSR™ Plus, Catalog #05825, or TeSR™-E8™, Catalog #05990)

Protocol

Before You Begin: Use sterile technique when coating cultureware. Non-tissue culture-treated cultureware is required for use with Vitronectin XF™.

1. Thaw Vitronectin XF™ at room temperature (15 - 25°C).
2. Using a 50 mL polypropylene conical tube, dilute Vitronectin XF™ in CellAdhere™ Dilution Buffer to reach a final concentration of 10 µg/mL (i.e. use 40 µL of Vitronectin XF™ per mL of CellAdhere™ Dilution Buffer).
3. Gently mix the diluted Vitronectin XF™. Do not vortex.
4. Immediately use the diluted Vitronectin XF™ solution to coat non-tissue culture-treated cultureware. See Table 1 for recommended coating volumes.
   
   

   Table 1. Recommended Volumes for Coating Cultureware

   Cultureware

   Volume of Diluted Matrix

   6-well plate

   1 mL/well

   100 mm dish

   6 mL/dish

   T-25 cm² flask

   3 mL/flask

   T-75 cm² flask

   8 mL/flask
5. Gently rock the cultureware back and forth to spread the solution evenly across the surface.
   
   
   Note: If the cultureware surface is not fully coated by the Vitronectin XF™ solution, it should not be used for human ES or iPS cell culture.
6. Incubate at room temperature (15 - 25°C) for at least 1 hour before use. Do not let the Vitronectin XF™ solution evaporate.
   
   
   Note: If not used immediately, the cultureware must be sealed to prevent evaporation of the Vitronectin XF™ solution (e.g. with Parafilm®) and can be stored at 2 - 8°C for up to 1 week after coating. Allow stored coated cultureware to come to room temperature for 30 minutes before proceeding to step 7.
7. Gently tilt the cultureware onto one side and allow the excess Vitronectin XF™ solution to collect at the edge. Remove the excess solution using a serological pipette or by aspiration. Ensure that the coated surface is not scratched.
8. Wash the cultureware once using CellAdhere™ Dilution Buffer (e.g. use 2 mL/well if using a 6-well plate).
9. Aspirate wash solution and add your desired cell culture medium (e.g. 2 mL/well if using a 6-well plate).