Balancing the Cryopreservation Equation
by Dr. Maria Thompson, BioCision VP of scientific affairs, [email protected]; Dr. Eric Kunkel, BioCision senior VP, research and development,
[email protected]; and Dr. Rolf Ehrhardt, BioCision president and chief executive officer, [email protected]
Abstract
A great deal of time, money
and energy has been spent on
optimizing cryopreservation
techniques. These efforts often
concentrate on improving freezing rates, freezing and storage
temperatures, storage vessels
and freezing media. In terms
of effectiveness in preserving
cell viability and function during cryopreservation, extensive
optimization of freezing alone
is misguided if cell thawing is
overlooked. The causes behind
post-thaw cell death have as
much to do with the mechanics of thawing as with freezing.
Although it has been acknowledged since the 1970s that thawing rate affects cell viability, to
date, little research has been carried out in this area, and there are few, if
any, automated temperature control solutions for the thawing portion of any given
cryopreservation protocol.
In this article, we will discuss the thawing arm of cryopreservation and the need
to strictly control temperature throughout
the process.
Optimizing Cell Survival
The goal of any cryopreservation
protocol is to preserve, as perfectly as possible, the structure and viability of living
cells and tissue in a state of suspended
animation. The rapid progress being made
today in regenerative medicine and biomedical research means there is constant
pressure for a reliable supply of donated
The Mechanics of Cell
Thawing
To understand the factors
at play during the freezing and
thawing of cells, we must first
review some of the basic physics
involved in the process.
blood, tissue, cells and other biospecimens.
Cryopreservation therefore provides an
important method of mitigating supply
and demand, in addition to its role in
protecting discrete cell populations from
genetic drift and differentiation. While the
ability to place cells and tissue in a temporary state of deep freeze is of enormous
benefit to the medical and biotechnological
industries, even state-of-the-art methods
take for granted that there will be a certain
amount of loss in viability and function
due to structural damage incurred during
freezing and thawing. So how does one go
about improving post-thaw cell survival?
Cells, and in particular stem cells, are
at their most vulnerable when transitioning from an active metabolic state to a
cryopreserved state and vice versa. This
process causes both mechanical and chemical stress, which can kill cells
outright, or trigger apoptosis
once cells are thawed [1]. Ultimately, the most important
factors impacting survival
of a frozen and thawed cell
are the cooling and thawing rates; these parameters
will affect solute distribution
and subsequently, ice crystal
formation and growth [2]. In
light of this, we need to ensure that both freezing and
thawing protocols are optimized.
“A quick review of the scientific
literature shows that in
contrast to the thousands of
papers devoted to optimizing
freezing techniques, there
are surprisingly few on the
biophysical aspects of thawing.”
Cold Facts | December 2014 | Volume 30 Number 6
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Initial cooling of a cell
slows metabolic processes with
little effect on cellular integrity.
Once cooling proceeds below the
freezing point of water, however,
the formation of extracellular
ice crystals effectively increases
the local solute concentration
around the cells. Water moves
out of the cells by osmosis, beginning the process of dehydration and cell shrinkage. Organic
solvents and other cryopreservatives are added to cryopreservation media
to lower the freezing point and depress the
formation of large ice crystals while the cell
dehydrates. If the cooling rate is too rapid,
intracellular ice crystals can form, damaging the cell before it can dehydrate. Ideally,
the cooling rate should be slow enough to
prevent intracellular ice formation, but fast
enough to prevent serious dehydration affects.
During cell thawing, the process of dehydration is reversed. As cells warm, any
ice present within the extracellular space
melts and the extracellular environment
becomes hypotonic; water rushes into the
cell, leading to cell swelling and/or bursting. Additionally, if the thawing rate is too
slow or the temperature within the sample
is uneven (as often happens with current
thawing protocols), the influx of water into
the cells can result in the growth and fusion of intracellular ice crystals.
While the use of cryopreservatives has
undoubtedly increased cell survival rates,
more recent research on the nucleation of
ice crystals has made it clear that one of
the major causes of cell death is actually
ice recrystallization during thawing rather
than ice crystal formation during freezing
[4]. And while many studies have identified the optimal cooling rate for preventing
cryoinjury in various cell types [3], little is
www.cryogenicsociety.org
known about the optimal thawing rates for
these same cell types.
stored in freezers that are accessed repeatedly, during transport from one facility to
another or during prolonged exposure to
room temperature air, as when storage box
racks are removed from the storage tank to
access other samples. When cells are ready
to be thawed for use, proper care must be
taken to safeguard them from variations in
temperature; an ideal protocol will thaw
cells swiftly and at a controlled rate.
A quick review of the scientific literature shows that in contrast to the thousands
of papers devoted to optimizing freezing
techniques, there are surprisingly few on
the biophysical aspects of thawing. We do
know that actively thawing cells at higher
temperatures, and therefore faster thawing rates, results in higher post thaw cell
viability, presumably by melting ice crystals faster than they can recrystallize [3, 4].
Faster thawing also helps protect cells from
cytotoxic effects associated with high concentrations of many cryoprotective agents,
including DMSO and glycerol [5, 6].
The number of freeze-thaw cycles, as
well as the absolute temperature a cell is
exposed to, will also affect whether a cell
survives the cryopreservation process. Basically, the greater the number of times cells
are partially thawed and then re-frozen, the
greater the risk of ice recrystallization [8].
Even if cell membranes are not disrupted,
repeated stress can trigger apoptotic pathways, decreasing the chances that cells will
survive or retain their optimal function
The Need for Standardization
Figure 1: BioCision’s ThawSTAR is one of the
first devices on the market specifically designed to standardize the cell thawing process
by precisely controlling the rate of thawing.
post-thaw. [1] Care must be taken to ensure
that cryopreserved cells are not exposed to
elevated temperatures during the freezing
process or in transport to liquid nitrogen
storage. Cells are most susceptible when
The current accepted methodology
for thawing cells is far from standardized;
most often, cells are thawed manually in a
37°C water bath or by rolling vials between
hands. The “end point” of cell thawing is
subjective and relies on an individual researcher’s observation, with little regard to
the optimal temperature and thaw rate for
a given cell type. This can introduce significant variability into the results that are
obtained. Additionally, thawing cells in a
water bath carries a high level of risk for
sample contamination. In a manufacturing
or clinical setting, most good manufacturing practice protocols require that water
Continued on page 11
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The Cryopreservation Equation... Continued from page 9
baths be emptied and cleaned after every use,
which is a time-consuming and costly procedure. Driven in part by the FDA’s oversight of
cell therapy, there is now an emerging need to
develop a standardized cell thawing device that
circumvents the need for a water bath.
of cryopreserved cord blood: the impact on graft quality, recipient safety, and transplantation outcomes.” Transfusion. Jun. 2014.
8. J. P. Erinjeri, T. W. Clark. “Cryoablation: Mechanism of Action and Devices. Journal
of Vascular and Interventional Radiology.” 21(8): pages S187-S191. August 2010.
Temperature Control Technology
Having the right temperature control technology is the key to optimizing cryopreservation.
Whereas modern technology offers several different solutions aimed at controlling and standardizing the cell freezing process (controlled-rate
freezers and bench-top passive freezing devices,
to name a few), there are few such solutions on the
thawing side of the equation, a gap that needs to
be addressed. Only now are we beginning to see
automated thawing devices (see Figure 1 on page
9) enter the cryopreservation arena. With any
luck, using devices of this type will soon become
standard practice. Standardizing temperature
control will no doubt have a vast positive impact
on the biotherapeutic industry. Going forward,
we hope to see increased awareness of the importance of optimizing the cell thawing process.
Certainly the time has come to reap the benefits of
balancing the cryopreservation equation.
References
1. A. Bissoyi, B. Nayak, K. Pramanik, S. K. Sarangi.
“Targeting cryopreservation-induced cell death: a
review.” Biopreserv Biobank. 2014 Feb.;12(1): pages
23-34. doi: 10.1089/bio.2013.0032. PubMed PMID:
24620767.
2. P. Mazur, S. P. Leibo, J. Farrant, E. H. Y. Chu, M. G.
Hanna, and L. H. Smith. “Interactions of cooling
rate, warming rate, and protective additive on the
survival of frozen mammalian cells.” Ciba Foundation Symposium on the Frozen Cell. 1970.
3. K. J. Chua, and S. K. Chou. “On the study of the
freeze-thaw thermal process of a biological system.” Applied Thermal Engineering 29: pages
3,696-3,709. 2009.
4. R. C. Deller RC, M. Vatish, D. A. Mitchell, and M.I.
Gibson. “Synthetic polymers enable non-vitreous
cellular cryopreservation by reducing ice crystal
growth during thawing.” Nature Communications
5:3244. Feb. 2014.
5. W. J. Armitage WJ and P. Mazur. “Toxic and osmotic effects of glycerol on human granulocytes.”
American Journal of Physiology 247(5 pt 1): pages
C382-389. 1984.
6. T. A. Grein, D. Freimark, C. Weber, K. Hudel, C.
Wallrapp, P. Czermak. “Alternatives to dimethylsulfoxide for serum-free cryopreservation of human
mesenchymal stem cells.” International Journal of
Artificial Organs 33(6): pages 370-380. 2010.
7. S. Akel, D. Regan, D. Wall, L. Petz, and J. McCullough. “Current thawing and infusion practice
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