Intracellular Ice Formation in Adipose Derived Adult Stem Cells (asc) in the Presence of Polyvinylpyrrolidone (pvp)

The main objective of this work was to optimize the serum free, DMSO free cryopreservation protocol for adipose derived adult stem cells (ASCs), suspended in polyvinylpyrrolidone (PVP) solution, which would help to avoid intracellular ice formation (IIF) – a well known cause of cell death during cryopreservation [1]. Passage 1 (P1) ASCs were cultured in our lab, washed and suspended in a 10% w/v solution of PVP in 1x Phosphate Buffered Saline (PBS). The freezing experiments were carried out using a fluorescence microscope (Nikon Eclipse E600) equipped with a cryostage using two different protocols – protocol 1 and protocol 2 (details in ‘materials and methods section) at different freezing rates viz. 1, 5, 10, 20 & 40 °C/min. Occurrence of IIF was determined by observation of ‘flashing’ (sudden blackening of the cells during the freezing ramp). Protocol 1 yielded nearly 100% IIF for all the freezing rates studied whereas protocol 2 yielded 0, 40, 47, 67 & 100% IIF for the above mentioned freezing rates respectively. The results indicated that employing protocol 2 for serum free cryopreservation may yield a better postthaw viability for P1 ASCs.