Cryopreservation and freeze-drying can be used to preserve cells or tissues for prolonged periods. Vitrification, or ice-free cryopreservation, is an alternative to cryopreservation that enables cooling cells to cryogenic temperatures in the absence of ice. The processing pathways involved in (ice-free) cryopreservation and freeze-drying of cells and tissues, however, can be very damaging. In this chapter, we describe the principles underlying preservation of cells for which freezing and drying are normally lethal processes as well as for cells that are able to survive in a reversible state of suspended animation. Freezing results in solution effects injury and/or intracellular ice formation, whereas drying results in removal of (non-freezable) water normally bound to biomolecules, which is generally more damaging. Cryopreservation and freeze-drying require different types of protective agents. Different mechanistic modes of action of cryoprotective and lyoprotective agents are described including minimizing ice formation, preferential exclusion, water replacement, and vitrification. Furthermore, it is discussed how protective agents can be introduced into cells avoiding damage due to too large cell volume excursions, and how knowledge of cell-specific membrane permeability properties in various temperature regimes can be used to rationally design (ice-free) cryopreservation and freeze-drying protocols.
Keywords: Anhydrobiosis; Cryopreservation; Cryoprotectants; Lyoprotectants; Membrane permeability; Membrane phase behavior; Preferential exclusion theory; Vitrification; Water replacement theory.