Biomedical Engineering Reference
In-Depth Information
better cell protection during cryopreservation. Cryoinjuries can be manifested as
cell lesions, caused by the reduction of selected functions to the complete cell
destruction and/or cytolysis [ 4- 9 ]. Initially it has been considered that cryoinjuries
are derived exclusively from the effect of extracellular ice crystals. In this sense, it
has been proposed that the use of an adequately high freezing rate will help to
avoid crystallization. However, a hypothetical cooling rapidity, so high that it
would not cause cell cryoinjury, could not be achieved in practice due to thermo-
dynamic limitations of heat transfer. In addition, during the “ultra-rapid” cooling,
despite the expectations, complete cell destruction occurred [ 4- 10 ] . At present it is
considered that cryoinjuries result from the extensive volume reduction (cellular
dehydration) and/or massive intracellular ice crystallization (mechanical damage)
[ 6, 22, 31- 33 ]. Although independent, these mechanisms can also act together. The
first event is expressed primarily at low-rate (£10°C/min) freezing and the second
one in high-rate (³ 10°C/min) freezing [ 10, 22 ]. In the course of low-rate freezing,
extracellular crystallization occurs initially. These ice crystals do not cause
mechanical cell damage (by membrane penetration) despite their physical pres-
ence [ 4- 7, 10 ]. However, crystallization produces a progressive rise in osmotic
pressure in extracellular area. Thus, extensive differences in the osmotic gradient
(extracellular vs. intracellular) are formed, resulting in intensive intracellular water
efflux. Consequently, cells become dehydrated (process known as “solution
effect”). Dehydration and hypertonicity cause cell volume reduction, malforma-
tions, and finally cytolysis [ 4- 9, 20- 22 ]. At high-rate (rapid) freezing, the osmotic
gradient has no time to develop, and due to that dehydration and cell volume reduc-
tion are minimal. However, intracellular ice crystal formation/growth and cell
destruction have the most critical effect. The process is named “mechanical cell
damage” [ 10, 22 ]. The quantity of “free” intracellular water can be increased by
the release of “bound” water. This process is called “water desorption.” The degree
of mechanical cell damage is related to the total intracellular ice mass and the size
of ice crystals [ 4- 10 ]. Cryoinjuries may occur not only at freezing but also due to
ice “recrystallization” and/or “dilution shock” during thawing. Small ice crystals
will either form crystal “agglomerates” or they will amplify their mass by “recrys-
tallization.” During intracellular “recrystallization,” mechanical cell damage
occurs, while in the state of their extracellular occurrence cell dehydration will be
expressed [ 4- 10 ] .
Inversely, rapid ice thawing causes a considerable increase in the extracellular
water mass and a subsequent osmotic pressure decrease. Due to that, but also due
to cryoprotectant slow efflux from cells, an extensive water influx into the cells
(“dilution shock” or “cell swelling”) will occur. Otherwise, cells will be more sen-
sitive to the increase, than to the decrease of its volume, and thus, they will be
destroyed [ 10, 22 ] .
Therefore, determination of an optimal freezing rate (specific for each cell type
and cryobiosystem) should be seriously considered. It is the speed of cooling which
has to be high enough to prevent cell dehydration and adequately low to make efflux
of water from the cell, possible. It would be ideal to find a cooling rate just less than
Search WWH ::




Custom Search