exposure to full-strength vitrification solution results in further
loading with CPAs but more importantly serves to dehydrate the
sample prior to cooling. This is done to increase the intracellular
solute concentration while decreasing the probability of intracellular ice formation. Timing of this step is critical to avoid CPA
toxicity. During thawing/warming, the CPA concentration is
decreased in serial steps, and sugars are used as osmotic buffer to
prevent excessive swelling of the cells (see Fig. 5).
5.3 Loading Cells
with Lyoprotective
Agents
Prior to (freeze-)drying, lyoprotective agents, such as trehalose,
need to be introduced into mammalian cells for intracellular protection. One of the major hurdles here is to overcome the inherent
impermeability of cellular membranes to disaccharides (see Fig. 4b).
Various approaches have been used to introduce trehalose into cells
in order to enhance freezing and/or drying survival. Trehalose
loading approaches include the application of genetically engineered pore-forming proteins [66], synthetic biopolymers which
interact with membranes [67], cell-penetrating peptides [68], engineered lipophilic membrane-permeable derivatives of trehalose
[69], nanoparticle-mediated intracellular delivery of encapsulated
trehalose [70], and intracellular delivery using thermally responsive
nanocapsules [71]. Further methods that can be employed to
facilitate trehalose uptake include electro-permeabilization [72],
osmotic shock [73], fluid-phase endocytosis [74], and thermal
cycling while passing membrane phase transitions at suprazero
temperatures [75] or at subzero temperatures [76].
For a variety of cell types, it has been shown that when they are
frozen in the presence of (high concentrations of) extracellular
trehalose, this results in surprisingly high post-thaw survival when
using optimized cooling rates [54, 76–78]. In these studies, no
measures were taken to introduce trehalose into the cells prior to
freezing. It appeared that trehalose enters into the cells during
freezing and/or thawing due to a combination of freezing-induced
osmotic forces and fluid-to-gel membrane phase transitions
[76]. Freezing-induced membrane phase transitions result in temporary membrane imperfections, which facilitate leakage of intracellular components and uptake of solutes along concentration
gradients (see Fig. 3d). Freezing-mediated loading of cells with
disaccharides has been shown to be beneficial for freeze-drying of
cells and stabilizes intracellular biomolecular structures (i.e.,
DNA/nuclei) during dry storage [54, 79].
5.4 Loading Tissues
with Protective Agents
When a tissue is immersed in a solution containing cryo- or lyoprotective agents, osmotic and concentration gradient driving
forces result in solute and water transport fluxes until an equilibrium state is reached. The dimensions over which mass transport
takes place during protectant loading are larger compared to those
during CPA loading of cells, and the outer and inner regions may
16
Willem F. Wolkers and Harrie ¨ tte Oldenhof
loading with CPAs but more importantly serves to dehydrate the
sample prior to cooling. This is done to increase the intracellular
solute concentration while decreasing the probability of intracellular ice formation. Timing of this step is critical to avoid CPA
toxicity. During thawing/warming, the CPA concentration is
decreased in serial steps, and sugars are used as osmotic buffer to
prevent excessive swelling of the cells (see Fig. 5).
5.3 Loading Cells
with Lyoprotective
Agents
Prior to (freeze-)drying, lyoprotective agents, such as trehalose,
need to be introduced into mammalian cells for intracellular protection. One of the major hurdles here is to overcome the inherent
impermeability of cellular membranes to disaccharides (see Fig. 4b).
Various approaches have been used to introduce trehalose into cells
in order to enhance freezing and/or drying survival. Trehalose
loading approaches include the application of genetically engineered pore-forming proteins [66], synthetic biopolymers which
interact with membranes [67], cell-penetrating peptides [68], engineered lipophilic membrane-permeable derivatives of trehalose
[69], nanoparticle-mediated intracellular delivery of encapsulated
trehalose [70], and intracellular delivery using thermally responsive
nanocapsules [71]. Further methods that can be employed to
facilitate trehalose uptake include electro-permeabilization [72],
osmotic shock [73], fluid-phase endocytosis [74], and thermal
cycling while passing membrane phase transitions at suprazero
temperatures [75] or at subzero temperatures [76].
For a variety of cell types, it has been shown that when they are
frozen in the presence of (high concentrations of) extracellular
trehalose, this results in surprisingly high post-thaw survival when
using optimized cooling rates [54, 76–78]. In these studies, no
measures were taken to introduce trehalose into the cells prior to
freezing. It appeared that trehalose enters into the cells during
freezing and/or thawing due to a combination of freezing-induced
osmotic forces and fluid-to-gel membrane phase transitions
[76]. Freezing-induced membrane phase transitions result in temporary membrane imperfections, which facilitate leakage of intracellular components and uptake of solutes along concentration
gradients (see Fig. 3d). Freezing-mediated loading of cells with
disaccharides has been shown to be beneficial for freeze-drying of
cells and stabilizes intracellular biomolecular structures (i.e.,
DNA/nuclei) during dry storage [54, 79].
5.4 Loading Tissues
with Protective Agents
When a tissue is immersed in a solution containing cryo- or lyoprotective agents, osmotic and concentration gradient driving
forces result in solute and water transport fluxes until an equilibrium state is reached. The dimensions over which mass transport
takes place during protectant loading are larger compared to those
during CPA loading of cells, and the outer and inner regions may
16
Willem F. Wolkers and Harrie ¨ tte Oldenhof