Cryopreservation
Optimising the cryopreservation process
Current preservation protocols rely on two different approaches:
conventional slow cooling/rapid warming and vitrification.
Relatively low survival of hESC is often reported both in the
literature and anecdotally for both techniques. An optimised
cryopreservation protocol is essential; especially in delivering
clinical grade cells where cell numbers may well be critical. In
conjunction with the Medical Cryobiology Unit and the Department of
Biology at the University of York, a PhD project is underway which
involves a methodological approach to the problem. The aim of this
approach is to separate the multiplicity of damaging factors, so
that each can be addressed in turn free of confounding effects of
the others. Two representative stem cell lines (Shef 3 and RH1)
were chosen to represent lines generally passaged by the two
current methodologies (so-called "cut-and-paste" and enzymic
disaggregation) as well as the embryonal carcinoma cell line
2102Ep.
During cryopreservation, cells may be subjected to a number of
damaging events which begin with the exposure of the cells to the
cryoprotective agent. Two cryoprotectants were chosen for this
study: dimethyl sulphoxide (DMSO), used in most conventional
freezing protocols, and propylene glycol, often used in combination
with DMSO in vitrification solutions. Damage caused by the
cryoprotectant may occur as a result of osmotic forces during its
addition and elution, as the cells shrink or swell in response to
the osmotic gradient, or through intrinsic toxic effects exerted by
the cryoprotectants; effects that are time and temperature
depended. In order to model safe addition and elution protocols, so
that concentration dependent toxicity can be investigated, certain
biophysical parameters of the cell membrane were first elucidated.
These parameters: non-osmotic volume of the cell (Vb), hydraulic
conductivity (L p) and solute permeability (P s) have been
calculated for all three cell lines and both cryoprotectants at two
temperatures.
In conjunction with experiments to determine the limits of
shrinkage and swelling that the cells will tolerate without damage,
the values obtained have been used in a two-parameter mathematical
model to develop addition and elution protocols which can be used
to introduce and remove the cryoprotectants without causing osmotic
damage. The optimised protocols are now being used to determine the
intrinsic toxicity of the cryoprotectants. Once the limits of
toxicity are known the project will then move to the final phase
where the damaging effect of cooling will be investigated on cells
exposed to safe levels of cryoprotectant, introduced through
optimised addition and elution protocols.