Biomedical Engineering Reference
In-Depth Information
Cryoprotectants have other mechanisms of action, as well. The protective effect
of penetrating cryoprotectants is obtained also due to their colligate effect, i.e.,
capacity for water binding [ 5- 10 ]. Glycerol and DMSO are the first-class acceptors
of hydrogen bonds; accordingly they can strongly bind a high amount of water and
they have a high cryoprotective capacity. Thus, cells can be stored at below freezing
temperatures without excessive intracellular ice crystal formation and extreme cel-
lular dehydration [ 10, 29- 33 ]. Although the cryoprotectant penetration (across the
cell membrane) ability is critical for cell protection, it can be obtained by rapid
(DMSO) and slow penetrating glycerol, as well as with nonpenetrating (HES) com-
pounds. The temperature of cell is exposed to the cryoprotectant influence on the
penetration rate also [ 22, 27- 32 ].The main nonpenetrating cryoprotectant is HES, a
compound initially used (together with DMSO) for granulocyte freezing. HES has
a higher molecular weight than DMSO and predominantly, it acts extracellularly at
the time of low-rate freezing. There are reports showing that it is possible to cryo-
preserve different blood cells with HES and DMSO using controlled-rate or uncon-
trolled-rate freezing. Cells frozen by these methods have adequate postthaw recovery
and viability [ 14, 15, 24- 26, 31- 33 ] .
In summary, cryoprotectants can express protective effect by the reduction of cell
dehydration as well as by decreasing the intensity of intracellular crystallization.
However, they cannot protect cells from already existing excessive dehydration or
from the effect of previously formed intracellular ice crystals.
Stem Cell Cryopreservation Practice
The completion of SC transplant requires both efficient collection (by aspirations
from bone marrow or apheresis from peripheral/cord blood) and cryopreservation
techniques for obtaining an adequate cell yield and recovery. For SC cryopreserva-
tion, there are several well-known protocols, using primarily DMSO in autologous
plasma, in various concentrations [ 11, 32- 36 ]. Most authors suggest that the opti-
mal cooling rate during cryopreservation is 1°C/min, while according to others it is
2-3°C/min [ 17, 36, 44, 45 ]. The transition from liquid to solid phase is also critical
(because of releasing of the specific fusion heat), since a considerable reduction in
cell viability has been observed when this period is prolonged. Thus, the optimal
freezing rate for SCs has been shown to be 1°C/min, with an elevated cooling rapid-
ity during liquid to solid phase transition (from 0°C to −10°C) period [ 11 ] . Finally,
there is data showing that uncontrolled-rate freezing is also useful in SC cryopreser-
vation [ 13- 15, 24- 26 ]. However, this technique produces an “unbalanced” or
“changeable” cooling rate (around or over 3°C/min). In addition, the freezing bag
or canister configuration as well as the volume of cell concentrate are critical param-
eters, which can radically modify the freezing velocity. In practice, bone marrow SC
cryopreservation consists of the following steps: (a) aspirate processing (red blood
cell and plasma reduction), equilibration (cell exposure to cryoprotectant), and
freezing; (b) cell storage at −90±5°C (mechanical freezer), at temperature from
Search WWH ::




Custom Search