Biomedical Engineering Reference
In-Depth Information
to whether the ice or elevated salt concentration is the cause for damage during the
progressive freezing of cells. The effect of the cooling rate on water transport during
progressive cooling was demonstrated by Mazur et al. [
105
] and they correlated this
with cell survival. This eventually leads to a “two factor hypothesis,” which states
that solute damage occurs at low cooling rates where extracellular ice formation
is innocuous to the cells. On the contrary, at high cooling rates, intracellular ice
formation is generally lethal. Cryoprotectant is correlated with an optimum cooling
rate since there is a dramatic variation in the membrane permeability of each
cell type.
The issues surrounding the cryopreservation of multicellular systems, such as
tissues and organs, are infinitely more complex given that many cell types exist and
each will differ in the requirements for optimal preservation. The most considerable
obstacle to the cryopreservation of multicellular systems is likely to be extracellular
ice formation, which leads to the focus on the use of various cryoprotectants that
possess the ability to regulate the formation of extracellular ice. Thus, effective
cryopreservation of such systems would be expected to occur through an “ice-free”
cooling or vitrification process even though this necessitates a high concentration of
cryoprotectant. The issue of toxicity arises due to the greatly limited concentration
that a tissue can tolerate before freezing. In addition, an osmotic imbalance is
inevitable as cryoprotectants penetrate the cell membrane more slowly than water.
Thus, cell volume must be regulated carefully during the addition and removal of
cryoprotectants.
Vitrification is a common technique employed for the freezing of cells and
tissues to avoid cell damage from ice formation. During this process, the solution
transforms into a “glass-like” solid enabling the sample to be frozen under “ice-
free” conditions. Despite the attractive features of vitrification, it does possess some
limitations. For instance, when dealing with larger volumes the heat transfer in cells,
tissues, and organs does not permit vitrification without the risk of crystallization.
Consequently, a slow freezing process (0.5-100
ı
C/min cooling rate) is often applied
for preservation of large volumes. However, in the vitrification process, a very
rapid cooling rate (24-130,000
ı
C/min [
106
]) is applied, resulting in a glassy or
vitreous state, which is dependent on the concentration, viscosity, volume, and
cooling rate of the process. Although this high cooling rate minimizes cellular
damage, recrystallization can still occur during warming. To avoid the risk of
recrystallization, rapid and uniform warming can be achieved using microwaves
[
107
-
109
].
Two problems unique to vitrification are encountered when using the methodolo-
gies outlined earlier. First, the necessary cryoprotectant concentration is very high
and is sometimes too toxic for the cell. Although the process is feasible with lower
concentrations of cryoprotectants, a higher cooling rate must be used to achieve
a vitrified state. Second, some fractionation of the glassy state still occurs. The
extent to which this occurs is dependent on the volume and the cooling rate and
such relationships have been examined in detail [
106
]. Vitrification works well with
small volumes (i.e., <1 L) even though it is not useful for large volumes. However,
