4 or pH 7.05, for 2 or 24 h. The cells incubated for 2 h were subsequently washed with DPBS and incubated for 22 h in growth media. An MTS assay was performed at the 24 h mark

according to the manufacturer’s instructions. SAOS-2 cells were seeded into 6-well tissue culture plastic plates (Corning, UK) at 105 cells/well. After 24 h, the cells were washed with DPBS (pH 7.4), then DPBS (pH 7.05), and were then incubated (37 °C with 5% CO2) in “incubation media”: serum-free media with 0.2 M trehalose, with or without PP-50 at different concentrations, and water (18.2 MΩ.cm, Milli-Q® filtered, Millipore, USA), at pH 7.05. Following incubation, the osmolarity of all solutions was adjusted to that of the incubation media using 10 × DPBS (PAA, UK) and/or water unless otherwise stated. After 2 h of incubation (37 °C with learn more 5% CO2), the cells were washed twice with DPBS, and trypsin/EDTA was added at 200 μl/well. After 15 min of incubation (37 °C with 5% CO2), 500 μl/well growth PS341 media was added and the cells were centrifuged at 350g for 5 min and resuspended in 150 μl of 0.2 M trehalose in FBS. Controls using un-incubated

cells were also prepared and resuspended in FBS (90%) and Me2SO (10%). All samples were transferred into cryovials (Greiner, UK), and transferred into an isopropanol freezing container (Nalgene, USA), then passively cooled in a −80 °C freezer overnight, before storage in vapour-phase liquid nitrogen for at least 48 h. The cells were subsequently thawed by immersing the cryovials in a 37 °C water-bath, after which 850 μl/cryovial of growth media were slowly added. SPTLC1 After centrifugation, the cells were resuspended

in growth media, and added to the wells of 96-well plates (100 μl/well). Non-frozen SAOS-2 cells were seeded into the plates at 5000 cells/well. After 4 h of incubation (37 °C with 5% CO2), the media was changed to growth media of normal osmolarity. MTS assays were subsequently performed at 24, 48 and 72 h, according to the manufacturer’s instructions. equation(1) td=(t2-t1)ln2lnN(t2)N(t1) equation(2) N(0)=N(24)224tdThe number of metabolically active cells was found using a standard curve. The doubling times, td, were calculated using Eq. (1), where t1 and t2 represent the time at time-points 1 and 2, respectively, and N(t1) and N(t2) represent the number of cells at time-points 1 and 2, respectively. The numbers of proliferative cells immediately post-thaw were estimated using Eq. (2). SAOS-2 cells were seeded into 25 cm2 tissue culture flasks (Corning, UK) at 5 × 105 cells/flask. After 24 h, the cells were frozen as described above, scaling volumes appropriately for the growth area. The freezing protocols used were; 0.2 M trehalose (additional 133 mOsm/l) with or without 25 μg/ml PP-50, and the Me2SO control. Following thawing of the cells, using a 37 °C water-bath, an Annexin V/PI flow cytometry assay was performed.