Cell Culture – Freezing, Thawing, and Adapting Cells

Each time a new cell line has been completed (eg, stable selection, or receive a new cell line, or after adaptation in different media) freeze away several bottles of stock

  1. Growing the cells to confluency in plates or flasks
  2. For the freezing media, the use of 20% FBS, 10% DMSO DMEM media
  3. Keep frozen media on the ice before resuspending cells
  4. Pre-chilled tubes used for freezing
  5. Put the tube with the cells in containers slow freezing in the freezer overnight to 80 degrees
  6. The next day, transfer to liquid nitrogen
  7. Wear goggles or face protection when using liquid nitrogen tank

thawing cells

  1. Thaw the cells quickly in a water bath of 37 degrees to prevent them from dying (change tubes in both directions in a water bath)
  2. Pour into a tube with regular media and centrifuge bottom of the cell
  3. For the stable cell lines, growing in regular media first day, and a change to the selection media the next day

Cells adapt to the new conditions
(For example, suspension culture, new media)

  1. Split the cell divides twice the concentration of a lower dilution (possibly even 1: 2 split)
  2. When centrifuging the cell, use only about 500x g for 5 minutes

To convert from g to rpm using this formula:
((G = 118 x 10-7 x r x n2))
where g = relative centrifugal force
r = rotating radius in cm
n = rpm (revolutions per minute)