Cell culture in Mebiol^® flask:

1. Open package in sterile environment and add 10mL of culture media directly to Mebiol^® flask.
2. Close flask and lay in refrigerator (2-10 °C) for 3 hours to allow Mebiol® to absorb media.
3. Periodically shake flask gently to help dissolve the Mebiol® into the media. Make take up to a day for complete dissolution (maintain at cold temperature).
4. After dissolving, allow to sit in refrigerator until bubbles are eliminated.
5. Add cells/tissues into Mebiol^® at low temperature (2-10°C) and warm to 37°C to culture cells/tissues in a 3D hydrogel environment.
6. To recover the cells/tissues, cool the Mebiol® back down and dilute with 30-40 mL of cold saline or medium. This will take it out of gel phase. Suspended cells can be easily recovered via centrifugation.

Cell culture in 24-well plate:

1. Follow steps 1-4 above.
2. Before adding cells/tissues, remove desired amount of Mebiol^® and put into chilled centrifuge tube. Put remaining Mebiol® into refrigerator (still in original flask).
3. Add approximately 10⁵ cell/mL to chilled centrifuge tube and mix gently on ice.
4. Warm up 24-well plate and separate overlaying culture media to 37°C.
5. Add 200 µL of cold Mebiol^® suspension to the center of each well. Make sure Mebiol^® does not cover entire bottom of the well to make it easier to change overlaid medium in later step.
6. Overlay 400 µL of warmed culture media containing phenol red on Mebiol^® suspension. Culture at 37°C.
7. To recover cells, refrigerate at 2-8°C and dilute with 400 µL of cold saline or medium. This will take Mebiol^® out of gel phase. Suspended cells can be easily recovered via centrifugation.

Do not re-sterilize to avoid deterioration. Add media promptly after opening, store in refrigeration and use within 1 month.