Cryopreservation of adherent cells in 96 well plates.

Cryopreservation of adherent cells in multiwell plates for subsequent use in high throughput screening would decouple plate manufacture from assay, allow panels of cells to be frozen on a single plate and enable quality control. Cryopreserved plates would allow long term studies to be carried out with the same cell population. Currently no assay-ready multiwell plates are commercially available for use with cryopreserved cells. The technical hurdles to successful cryopreservation in a plate format are the effects of the extreme supercooling (20 K) encountered in small liquid volumes (typically 100 μL per well) and the large well-to-well temperature variations. This study exploited controlled rate freezers - which cool by conduction to achieve temperature uniformity within all wells during cooling. We also developed a new consumable which ensured that ice nucleation occurs with mimimal supercooling (less than 3 K) in all wells of the plate. Multiwell plates containing SAOS-2 cells, showed a 1.65 + 0.03 fold increase in viability post thaw, using the new technology. In addition to use in high throughput screening applications, this approach may also be exploited to screen new cryoprotectants and mixtures of cryoprotectants in an efficient and rapid manner, examples will be presented.