Remove a small aliquot of cells (100–200 µL) and perform a cell count. Ideally the cell viability should be in excess of 90% in order to achieve a good recovery after freezing. Centrifuge the remaining culture at 150 g for 5 minutes. Re-suspend cells at a concentration of 2-4x10 6 cells per ml in freeze medium.
The extent of cell wall rupture can be controlled by freezing produce as quickly as possible. In rapid freezing, a large number of small ice crystals are formed. These small ice crystals produce less cell wall rupture than slow freezing which produces only a few large ice crystals.
4) Freeze cells. To allow water to move out of the cells before freezing, freeze cells slowly. This is accomplished using some type of cell freezing chamber. Pricey freezing chambers pulse in liquid N 2 periodically to control the freezing rate. Less expensive options include chambers that use room temperature isopropanol.
Lethal intracellular freezing can be avoided if cooling is slow enough to permit sufficient water to leave the cell during progressive freezing of the extracellular fluid. To minimize the growth of extracellular ice crystal growth and recrystallization, biomaterials such as alginates, polyvinyl alcohol or chitosan can be used to impede ice crystal growth along with traditional small molecule cryoprotectants.