Biomedical Engineering Reference
In-Depth Information
to avoid cytotoxicity and excessive volumic range excursions during equilibration
stage, whilst obtaining a completely unfrozen size-distributed cell population.
Basically, a system behaviour at varying CPA content similar to the one
reported in Fig.
16
for the case of -50C/min may be found also for the other
cooling rates considered in this work. Specifically, at -1C/min a very high CPA
concentration needs to be used to obtain a completely unfrozen size-distributed
cell population, whilst, if CPA is completely absent, an innocuous level of IIF is
reached in every size class of the cell population. Conversely, at -400C/min
a significant, lethal IIF for any cell is obtained without using CPA, whilst, with a
CPA content as low as 7 kosmol/m
3
, a completely unfrozen size-distributed cell
population results. Along these lines, high cooling rates need to be combined to
high CPA contents for reaching vitrification, whilst at low cooling rates cell via-
bility may be increased by adopting relatively low CPA concentrations. These
conclusions perfectly match the well known rule of thumb for a successful
cryopreservation protocol. Clearly, the values of the operative conditions needed
to optimise the process in terms of final cell viability strongly depend on the
osmotic characteristics and initial cell size distribution of the specific cell lineage
at hand.
4 Concluding Remarks and Future Directions
A novel theoretical interpretation of the freezing process for a suspension of cells
has been proposed. It is based on the PBM approach addressing the size distri-
bution of the cell population. The latter one is invoked to interpret and rationalise
the experimental evidence measured so far in a more comprehensive fashion than
the other theoretical analysis developed before.
After the initial experimental validation by direct comparison with suitable
experimental data taken from the literature, model investigations on system
behaviour at various operating conditions have been performed, i.e. different
CPA content and cooling rates. It is demonstrated that, cell survival due to intra-
cellular ice formation depends on the initial cell size distribution and its osmotic
parameters. At practicable operating conditions in terms of cooling rate and
cryo-protectant concentration, IIF may be lethal for the fraction of larger size
classes of the cell population whilst it may not reach a dangerous level for the
intermediate size class cells and it will not even take place for the smaller ones.
This has been also tested by removing the classic assumption of EIF under
thermodynamic equilibrium thus accounting for its dynamics. As such it may be
concluded that, when cryopreserving a cell suspension under practicable oper-
ating conditions the cell size distribution of the cell population and the dynamics
of EIF should be carefully taken into account, contrarily to what has been
classically assumed.
In the next future, the model approach developed so far will be adopted to
simulate in a comprehensive way a complete cyopreservation cycle by accounting
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