Biomedical Engineering Reference
In-Depth Information
Figure 7.12
Effect of cooling rate on cells.
to water and initial intracellular water concentration will determine the precise rate
of cooling. To reduce injury to cells during cryopreservation, cryoprotective agents
(CPA) have been developed to both permeate the cell and to displace water to pre-
vent intracellular ice crystal formation. CPAs are antifreeze components added to
the solutions in which the cells are being frozen. Polge et al. [5] successfully cryo-
preserved mammalian cells in 1949 using glycerol as a CPA. Another commonly
used CPA is dimethyl sulfoxide, which may not be appropriate for all cells, as it
could induce differentiation.
It is important to control the freezing as well as thawing environment in order
to maximize cell viability. Cryopreserved cells are extremely fragile and require gen-
tle handling. Cryopreserved cells should be thawed for use as rapidly as possible,
without letting the temperature exceed the physiological temperature range. Quick
thawing prevents recrystallization (the tendency of small ice crystals to increase in
size) of intracellular ice and minimizes the exposure time to high solute concentra-
tions. After thawing, the cells are transferred to a flask and a fresh medium is added
dropwise and slowly to prevent osmotic damage to cells. After adherent cells attach
and spread, the medium is changed to remove the CPA. A thawed culture may re-
quire a certain amount of time before usage in a specific application.
Steps for obtaining increased cell numbers have been developed. For most cells,
1
C/min is used, which is performed in a controlled rate freezer. First, adherent
cells are removed from the substrate by enzymatic treatment and resuspended as
a suspension at high cell densities (at least 106 cells/mL) in a medium containing
serum and a CPA. When glycerol is used, the cells should be allowed to rest in the
solution containing CPA for 30 minutes at room temperature prior to cooling to
ensure that the CPA permeates through the cell membrane without suffering from
osmotic shock. To avoid the possible effect of the osmotic pressure induced by a
sudden change of concentration of CPA, it might be effective to raise the concen-
tration of CPA gradually. The major steps in the cryopreservation process include:
°
1. Adding CPAs (at a concentration of 10% v/v) to cells/tissues before cooling;
2. Cooling the cells/tissues to a low temperature (e.g.,
196°C, liquid nitrogen
temperature at pressure of 1 atm) at which the cells/tissues are stored;
3. Warming the cells/tissues;
4. Removing the CPAs from the cells/tissues after thawing.
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