2-D Cell Growth on Hydrogels

This protocol describes how to make HyStem®-C hydrogels in a 96-well plate format for cell growth on the surface of the hydrogels. The protocol can easily be adapted for use with 6-, 12-, 24-, and 48-well plates. It can also be adapted for use with HyStem-HP hydrogel kits, merely substitute all instances of Glycosil with Heprasil.

• HyStem-C or HyStem-HP hydrogel kit
• One sterile 96-well plate

• 1. Prepare HyStem-C Hydrogel Kit components under aseptic conditions as directed by the instructions.
   
• 2. Mix equal volumes of Glycosil® and of Gelin-S®, either 1.0 mL each or 5.0 mL each depending on kit size.
  Pipette up and down thoroughly to mix. Note: If the HyStem® solutions are not well mixed, then the hydrogel surface may not be uniform. This can cause variation in how the cells attach and grow on the hydrogel.
   
• 3. When you are ready to pour the hydrogels, add one part Extralink® to four parts Glycosil + Gelin-S mix (typically 0.5 mL Extralink to 2.0 mL mixture, or 2.5 mL Extralink to 10 mL mixture depending on kit size). Pipette up and down thoroughly to mix. Note: Once the Extralink is added you have < 20 minutes before the hydrogel forms and it becomes impossible to uniformly pipette the solution.
   
• 4. Pipette 100 μL of hydrogel into each well. Place the lid on the 96-well plate.
  
  Note: Leftover solutions can be frozen at -20°C and are viable for ~2 weeks.
   
• 5. Allow the hydrogel to gel by placing the plate on a rocker for at least one hour with the lid on.
  
  Note: If the hydrogel is left for an extended period of time, it will dry out and form a film.
   
• 6. Prepare cells for use in 2-D cell culture as per standard procedures.
  Seeding density varies with cell type, but a typical range is 5,000 to 50,000 cells per well.
   
• 7. Once the hydrogels are solid, add 100 μL of cell slurry in media to each well on top of the hydrogel.
   
• 8. Place in the 37°C incubator with 5% CO2.

Changing Media

• 1. Carefully aspirate off the media.
  The hydrogel can easily be removed by the vacuum as well, so this must be done gently and carefully.
   
• 2. Pipette 100 μL media into each well.
  Try to avoid disrupting the gel.
   
• 3. Return the plate to the 37°C incubator with 5% CO2.