Biomedical Engineering Reference
In-Depth Information
system” (cryobiosystem) and determines data/facts applicable in cryopractice.
While the goal of the cell culturing is to keep cells in as near-normal conditions as
possible by supplying nutrients essential for their metabolism, cooling/freezing is
an effort to reduce the necessity of cells for energy. Precisely, after cooling, the need
of cells for energy production (ATP synthesis) and its consumption (for protein
synthesis, ion transport, and other biochemical activities) is decreased [
2
] .
Cryopreservation is beneficial when cells appear to be biologically, chemically, or
thermally unstable after liquid-state storage. Its primary purpose is to obtain both
better cell recovery and well postthaw viability. Thus, cryopreservation includes
specific approaches and techniques designed to extend “therapeutic shelf-life” of
the cells (prolonged storage time) and to obtain minimum thermal damage (cryoin-
jury) [
1
]. The use of cryobiology for isolated cell preservation began in 1949 with
the freezing of living animal sperm cells, using glycerol as a cryoprotectant [
3
] .
Afterward, glycerol and DMSO techniques were applied for cryopreservation of
different blood-derived cells [
3-
9
]. The basic goal of these initial cryoinvestigations
was to predict the cell response to freeze/thaw processes and cryoprotectant addi-
tion/removal. However, evaluation of cryobiological variables (biophysical, physi-
cochemical, and other parameters responsible for cryoinjury) as well as
standardization of practical aspects of cryopreservation is still a question of large
interest to researchers and practitioners [
10-
17
] .
Although SC cryopreservation is now in routine use, certain freezing aspects
should be revised to optimize specific cryobiosystem, i.e., to minimize the cryoin-
jury and maximize cell recovery [
10-
16
] . Microprocessor-restricted (controlled-
rate) freezing is a time-consuming process, which requires high-level technical
expertise. Uncontrolled-rate (“dump-freeze” without programmed cooling rate)
technique is less costly because it does not require a programmed freezing device.
However, there are data [
13,
18-
23
] that controlled-rate method is a high-class
alternative to uncontrolled-rate technique [
24-
26
] due to superior quantitative and
functional cell recovery. Finally, for obtaining an effective cryopreservation, besides
specific, i.e., optimized freezing method, the choice and use of appropriate cryopro-
tectant agent is required. At present, for SC and platelet freezing, DMSO and HES
are commonly used as cryoprotectants, although in different concentrations [
14-
16,
22-
33
]. The next part of text recapitulates the current knowledge on cell cryoinju-
ries and effects of cryoprotectants as well as the conceptual and practical aspects of
SC cryopreservation. In addition, recent activities in cryopractice, including our
results obtained by the controlled-rate system (with compensated fusion heat) vs.
uncontrolled-rate freezing (“dump-freezing” technique) in experimental and clini-
cal settings will be summarized.
Cryoinjury: Its Origin and Mechanisms
The object of fundamental cryoinvestigations is to determine physicochemical and
cryobiological attributes (including cell osmotic characteristics, water and cryo-
protectant permeability coefficients) in the course of freezing, as well as to obtain
