**3.5 Thawing methods**

Whether freezing is permitted (conventional cryopreservation) or prevented (vitrification), the CPA that has reached the internal compartments of a multicellular system must diffuse back through numerous membranes in the tissue, with each acting as a barrier. Therefore, optimal thawing and CPA removal procedures are also critical factors for cell/tissue survival after freezing (Bagchi et al., 2008). Earlier studies pointed out that consistent cooling and thawing rates (slow-freezing followed by slow-thawing, or fast-freezing followed by fast-thawing) can improve cell/tissue survival after cryopreservation (Whittingham et al., 1972). Moreover, extreme osmotic changes during CPA removal might damage the cells by extensive cell shrinkage or swelling associated with the rapid movement of water into the cell as compared to the slower movement of the CPA out of the cell. However, a limited amount of water replacement is needed to restore osmotic equilibrium and physiologic cell volume (Pegg, 2007).
