Fig 1.
The experimental cooling profiles used for cryopreservation of HepG2, CHO and MG63 cells.
1ml-filled cryovials were cooled in a controlled rate freezer at 1°C min-1. At selected temperatures between +4° and -100°C samples were plunged directly into liquid nitrogen (dashed lines), where they cooled at a rapid. uncontrolled rapid rate. A second experiment with endpoint temperatures between -38 and -50°C was also carried out.
Fig 2.
An example of a DSC trace, to which a second time derivative has been applied, used to measure Tg’i for a sample of HepG2 cells).
A trace from a cell suspension is shown in black, together one for the cryoprotectant solution without cells (grey). For the cell suspension a transition is seen at point A (black) corresponding to the intracellular glass transition Tg’i.
Fig 3.
Adherent cell count (A), and redox activity (B) of HepG2 (left), CHO (center) and MG63 cells (right) measured after 24h (grey), 48h (white) and 72h (black) of culture post-thaw following cryopreservation at a controlled rate of 1°C min-1 down to endpoint temperatures (ET) between +4° and -100°C before plunging into liquid nitrogen.
Grouping of ET for each analysis time point post-thaw is indicated by letters in the underneath tables, with distinct groups corresponding to P values < 0.05 and overlapping groups to 0.05 < P values < 0.10.
Fig 4.
Adherent cell count (A), and redox activity (B) of HepG2 (left), CHO (centre) and MG63 cells (right) measured after 24h (grey), 48h (white) and 72h (black) of culture post-thaw following cryopreservation at a controlled rate of 1°C min-1 to endpoints at 2-degree intervals between -38° and -50°C before plunging into liquid nitrogen.
Grouping of ET for each analysis time point post-thaw is indicated by letters in the underneath tables, with distinct groups corresponding to P values < 0.05 and overlapping groups to 0.05 < P values < 0.10.
Table 1.
Intracellular glass transition temperature for a range of cell types, determined by DSC analysis.
The temperature providing optimal cell recovery following cryopreservation of the nucleate, mammalian cell lines is presented as the safe temperature for transfer to long term storage.