MOJ Cell Science and Report
Ice-Free Cryopreservation by Vitrification
Abstract
Mini Review
Ice-free vitrification of biospecimens is an alternative cryopreservation strategy to
conventional preservation by freezing. Vitrification is the amorphous solidification
of a supercooled liquid. This state is achievable by adjusting the cryoprotectant
concentration and cooling rate to minimize nucleation and growth of ice crystals. The
cooled liquid is then converted to a glassy state, notice. Without ice crystal formation,
the biospecimens’ extracellular matrix and cell viability is often better preserved.
The decision to utilize an ice-free versus a freezing method for different types of
biospecimens depends on which method is easiest for the product, whether the
biospecimen will be washed before use, and whether an optimized method is already
available. Generally, cells and tissues can be preserved using ice-free vitrification
but isolated cells are easier to preserve using freezing methods because the cells are
exposed to less risk of cryoprotectant-induced cytotoxicity and the cryoprotectant
solutions are less viscous making the cells easier to handle. Samples such as tissue
biopsies, Islets of Langerhans and encapsulated cells can also be preserved using
either strategy, however the formation of ice during freezing may disrupt the
tissues and distort or break capsules. Ice-free vitrification has major advantages for
preservation of ovaries, heart valves, articular cartilage, and both natural and tissue
engineered blood vessels, protecting the extracellular matrix and cells. In the extreme
case of articular cartilage freezing results in less than 20% cell viability in contrast
with ≥80% after ice-free vitrification.
Volume 1 Issue 2 - 2014
Kelvin GM Brockbank1,2,3*, Zhenzhen
Chen1, Elizabeth D Greene1 and Lia H
Campbell1
Cell & Tissue Systems Inc., USA
Institute for Bioengineering and Bioscience, Georgia
Institute of Technology, USA
3
Department of Regenerative Medicine and Cell Biology,
Medical University of South Carolina, USA
1
2
*Corresponding author: Kelvin GM Brockbank,
Cell & Tissue Systems Inc., 2231 Technical Parkway,
Suite A, North Charleston, South Carolina, USA,
Tel: 843-722-6756; Fax: 843-722-6657; E-mail:
kbrockbank@celltissuesystems.com
Received: June 04, 2014 | Published: June 17, 2014
Keywords
Cryopreservation; Vitrification; Cells; Tissues; Tissue engineering; Regenerative
medicine; Tissue banking
Introduction
Long-term preservation is crucial as an enabling technology
for tissue banking for transplantation, regenerative medicine
products that contain living cells and biopsy samples.
Regenerative medicine reviews in particular have consistently
highlighted the need for preservation methods for product
development and commercialization. Strategic assessments of
regenerative medicine have provided three critical priorities
related to the need for better preservation methods [1-2]:
•
•
•
Assembling and Maintaining Complex Tissue
Improving Tissue Preservation and Storage and
Facilitating Effective Applications Development and
Commercialization
Cryopreservation by freezing may maintain cell viability and
adequate RNA [3], but histology and extracellular matrices are
poorly preserved [4-5] due to ice formation within the tissues
(Figure 1a).
Vitrification
An alternative strategy for cryopreservation of biological
materials is to promote vitrification by minimization of ice
formation (Figure 1b). Vitrification was first proposed for
cryobiology applications by Luyet [6]. Development of ice-free
tissue cryopreservation by vitrification in our work was initially
stimulated by the demonstration of large ice crystal domains
within frozen tissues (Figure 1a). The methods employed for
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the avoidance of ice in tissues were based on the early work
of Fahy et al. [7], Rall and Fahy [8], Rall [9]. Vitrification is the
amorphous solidification of a supercooled liquid. In ice-free
cryopreservation the formation of ice is prevented by the
presence of high concentrations of chemicals that interact
strongly with water and, therefore, prevent water molecules
from interacting to form ice. It has been shown that depressing
the homogeneous nucleation temperature until it equals the
glass-transition temperature permits vitrification of macroscopic
biological systems. Prevention of freezing means that the water
in a tissue remains liquid during cooling. As cooling proceeds,
however, the molecular motions in the liquid permeating the
tissue decrease. Eventually, an “arrested liquid” state known as a
glass is achieved. It is this conversion of a liquid into a glass that is
called vitrification (derived from vitri, the Greek word for glass).
A glass is a liquid that is too cold or viscous to flow. A vitrified
liquid is essentially a liquid in molecular stasis. Vitrification does
not have any of the biologically damaging effects associated with
freezing because no appreciable degradation occurs over time in
living matter trapped within a vitreous matrix.
The protocol most commonly used for viable ice-free
preservation by vitrification has been described in detail
[10]. Briefly, tissues in glass scintillation vials are gradually
infiltrated with VS55 at 4°C using cold cryoprotectant solutions
in six sequential, 15min steps of increasing cryoprotectant
concentration. After addition of the final vitrification solution,
the top of the vitrification solution is covered with 0.7ml of
2-methylbutane (isopentane, freezing point: -160°C, density:
MOJ Cell Sci Report 2014, 1(2): 00007
Ice-Free Cryopreservation by Vitrification
b)
Figure 1: Cryopreserved frozen (a) and vitrified (b) rabbit jugular
veins demonstrating the relative presence of ice in frozen tissue, and
absence of ice in vitrified tissue on the right after cryosubstitution
at -90°C. The ice/water in the samples was cryosubstituted with
methanol at -90°C and fixed with 1% osmium tetroxide as it
rewarmed so that the spaces occupied by ice (white spaces in tissue
on (a)) were preserved. Araldite embedded tissue sections were
stained with toluidine blue, original magnification 40X.
0.62) at 4°C to prevent direct air contact. Samples are then
cooled rapidly (~43°C/min) to -100°C by placing the sample
in the precooled (-135°C) 2-methylbutane bath (fast cooling),
followed by slow cooling (3°C/min) to -135°C in air in the storage
freezer, and then stored at -135°C in either a mechanical storage
freezer or ≥135°C near the top of vapor phase nitrogen freezer.
Vitrified tissues are rewarmed in a two-stage process including
slow warming (30°C/min) to -100°C and then rapid warming
(~225°C/min) to 4°C. After re- warming, the vitrification solution
is removed in a stepwise manner at 4°C in seven sequential,
15min steps [11].
Vitrification Versus Freezing
Vitrification and freezing in cryopreservation are not mutually
exclusive processes, the crystalline ice phase and vitreous phase
often coexist within a system. Many cells in research and some cell
therapy products, such as bone marrow, are simply cryopreserved
after addition of cryoprotectants to cells in suspension in
cryovials or bags by slow rate cooling, with or without induced
nucleation, and storage at -80°C or below -135°C. When cells are
cryopreserved in suspension using such freezing methods the cells
become vitrified in channels sandwiched between regions of ice.
This occurs because during freezing the concentration of solutes
in the unfrozen phase increases progressively until the point is
reached when the residual solution is sufficiently concentrated
to vitrify. Conventional freezing cryopreservation techniques are
optimized by designing using protocols that avoid intracellular
freezing. The cell contents vitrify due to the combined processes
of dehydration, cooling and the promotion of vitrification by cell
permeating cryoprotectants and intracellular macromolecules.
So technically, the cells are vitrified even though the method is
a freezing method. In contrast when cells that survive freezing
in suspension are placed on a substrate and cryopreserved using
the same simple freezing methods often very low cell viability
post-rewarming occurs (Figure 2).This is likely due to the cells
being immobilized on a substrate so that they cannot move away
from forming ice crystals. Further optimization of the freezing
method is required to maximize cell viability and attachment.
Similarly suspended chondrocytes are easy to cryopreserve
using freezing strategies but not when embedded in their natural
tissue matrices.
Cell suspensions can be preserved by ice-free vitrification
methods, but generally isolated cells are easier to preserve
using freezing methods. This is because of cell exposure to
high cryoprotectant concentrations under conditions that
promote cryoprotectant-induced cytotoxicity during ice-free
vitrification. Also the cryoprotectant formulations employed for
ice-free vitrification are very viscous making it more difficult
to handle cell suspensions during the cryoprotectant removal
steps. Cytotoxicity can be minimized by using the least toxic
cryoprotectant concentration possible that still promotes glass
formation [12,13] or by using addition and removal conditions
that minimize exposure of biological materials to cytotoxic
cryoprotectants. In the case of cells and tissues that are susceptible
to cryoprotectant cytotoxicity the final steps in addition and first
100
Cell Viability (%)
a)
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80
60
day 0
day 1
day 3/4
40
20
0
culture plate
cryovial
Figure 2: Cell viability after cryopreservation. Keratinocytes were
cryopreserved suspended in vials or adhered on culture plates
in 10% DMSO in RPMI culture medium. Metabolic activity was
measured immediately after thawing and for several days post thaw.
Cell viability (% untreated controls) was calculated as the mean
(±SEM) of 120 replicates.
Citation: Brockbank KGM, Chen Z, Greene ED, Campbell LH (2014) Ice-Free Cryopreservation by Vitrification. MOJ Cell Sci Report 1(2): 00007.
Ice-Free Cryopreservation by Vitrification
steps in removal of the cryoprotectants can be performed at subzero temperatures to minimize such concerns. Cryoprotectant
cytotoxicity can be used to advantage in situations where cell
viability is not required resulting in tissues that retain their
extracellular matrix with significantly reduced immunogenicity
[10,14-16].
Advantages of Vitrification
Ice-free vitrification offers several unique advantages over
traditional slow rate freezing with cryoprotectants. This state
is achievable by adjusting the cryoprotectant concentration and
cooling rate to minimize nucleation and growth of ice crystals.
The cooled liquid is then converted to a glassy state, notice.
Vitrification results in the elimination of ice crystal formation,
both within the cells and their extracellular matrix and in the
surrounding solution [17]. Without ice crystal formation, the
extracellular matrix and cell viability are better preserved
[5,18]. The protocols for vitrification are very simple [10]. The
concentration of cryoprotectants needed depends upon the
sample size and material properties of the tissue associated with
the cells. Small tissue samples, such as human embryos, may be
placed directly into the cryoprotectant and then plunged directly
into liquid nitrogen. Larger tissues need to be placed in increasing
concentrations of cryoprotectant, in a step wise manner, in order to
prevent cell damage due to osmotic effects. Higher concentrations
of cryoprotectants may be required by tissue samples because the
cooling rates achievable for larger samples are slower. Complete
vitrification should eliminate concerns for the known damaging
effects of intra- and extracellular crystallization that leads to loss
of cell viability, tissue morphology, extracellular matrix integrity
and RNA degradation.
Applications
Ice-free cryopreservation procedures have been developed
and shown to provide effective preservation for a number of
normal cell and tissue types, including monocytes, ova [19]
and early embryos, pancreatic islets, smooth muscle, blood
vessels, heart valves and cartilage [11,20]. The cell viability and
functional survival of these vitrified materials is usually 80% or
higher. Recent evidence indicates that ice-free cryopreservation
of embryos by vitrification is less traumatic than conventional
slow freezing and is, therefore, a more effective means of
cryopreserving the human embryo with excellent pregnancy
outcomes for ice-free vitrified embryos [21]. Support for using
ice-free vitrification to store cell and tissue therapy products and
tissue biopsies can be obtained from published studies reporting
retention of cell viability, phenotype and genotype employing
50-100µm diameter tissue samples in capillary tubes [22] and
250μL straws [23,24]. The small sample methods utilized are not
scalable for larger patient-derived tissue samples or engineered
tissues because the methods employed by these investigators
[22-24] used 40% (v/v) cryoprotective agent solutions. Such
low concentration solutions require very rapid cooling/warming
rates that are only possible with such very small sample
volumes. Ice formation on warming is of comparable or greater
importance than ice formation on cooling in determining survival
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of cryopreserved samples. The critical warming rates required
for ice-free warming of vitrified aqueous solutions of glycerol,
dimethyl sulfoxide, ethylene glycol, polyethylene glycol 200 and
sucrose are one to three orders of magnitude larger than critical
cooling rates [25].
Extensive studies of ice-free cryopreserved tissues have been
performed over more than a decade and the ice-free vitrified
tissues have been compared with conventionally, slow rate frozen
and fresh control tissues. Slow rate freezing with 10-15% DMSO
preserves at least some cell viability, whereas snap freezing with
or without cryoprotectants or products like All protect Tissue
Reagent (for DNA, RNA and protein preservation) and RNA later
(for RNA stabilization)retain tissue RNA but usually not cell
viability. Very rapid cooling techniques in which tissue samples
are plunged into coolant in contact with metal plates can result
in excellent ultrastructure preservation due to the absence of
ice formation. The last method involves ice-free vitrification
and was licensed for commercialization by Hitachi, the electron
microscope manufacturer, based upon technology acquired
from LifeCell Corporation [26]. Thus the concept of vitrification
strategies preserving cell/tissue structure and tissue matrix
components is well established. Other investigators developed
solutions and methods for rapid cooling of small samples of
cells in narrow tubes, such as straws, but most mammalian
tissues, for medical purposes, are too large for such containers
requiring larger volumes of more concentrated cryoprotectants.
The scale of these tissues also forces the use of slower cooling
rates (>40°C/min) simply because more rapid cooling rates are
not achievable. We have used a 55% (v/v) solution, VS55, that
consists of an 8.4M mixture of 1,2 propanediol, formamide, and
dimethyl sulfoxide in Euro Collins (EC) solution [8]. The protocol
is described in Khirabadi et al. [27-29] us Patents.
We have successfully applied vitrification strategies to a
variety of tissues tested to date from adult animals [5,15,1718,27-36] and some tissue engineered substitutes [37-38].
Vitrification also results in superior retention of extracellular
matrices compared with freezing methods [5,18] and excellent
post-transplant cell survival and tissue function in preserved
grafts [30,32].
Ice-free vitrification is outstanding for cryopreservation of
articular cartilage. Freezing studies using a variety of animal
articular cartilage models [39-42] and human cartilage biopsies
[43] have revealed no more than 20% chondrocyte viability
following conventional freezing methods of cryopreservation
employing low concentrations of either ME2SO or glycerol
as cryoprotectants. Chondrocytes in articular cartilage from
larger mammals were not adequately preserved using the VS55
cryoprotectant formulation [31] that was previously shown
to be effective for relatively thin rabbit cartilage [42]. Higher
cryoprotectant concentrations were then employed for relatively
thick porcine articular cartilage samples, without the bone
attached, resulting in significantly improved cell viability [31].
Increasing the cryoprotectant formulation to 83%, VS83, using
the same cryoprotectants while leaving the bone attached to the
cartilage, resulted in 80.5% viability immediately after rewarming
Citation: Brockbank KGM, Chen Z, Greene ED, Campbell LH (2014) Ice-Free Cryopreservation by Vitrification. MOJ Cell Sci Report 1(2): 00007.
Ice-Free Cryopreservation by Vitrification
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2014 Brockbank et al.
and viability returned to fresh control values by the second day
of tissue culture under physiological conditions (Table 1) [44].
Johma et al. [45] also demonstrated good viability when human
knee articular cartilage with bone attached was treated with 4
different cryoprotective agents for mathematically determined
periods of time at low temperatures. Cell viability, determined by
several different assays, was 75.4±12.1%.
solely the responsibility of the authors and does not necessarily
represent the official views of the National Institute of Biomedical
Imaging and Bioengineering or the National Institutes of Health.
Commercial use of protocols disclosed in this work is subject
to several issued US Patents (6,194,137; 6,596,531; 6,740,484;
7,157,222; 8,440,390) and International Patents (available upon
request).
We have observed solution fractures in ice-free vitrified
samples stored in vapor phase nitrogen [33] but only once
seen a macroscopic tissue fracture in a specimen (Brockbank,
unpublished observations). We believe this to be due to use
of relatively slow cooling rates as the samples go through the
glass transition phase change. However, we cannot rule out
the possible presence of microfractures being present in some
tissues. Thermo-mechanical stress is the response of a material
to volume changes associated with expansion or shrinkage of the
material during cooling. Significant temperature gradients arise
when a specimen of a significant size is cooled from outside in
and become increasingly severe as the cooling rate increases
resulting in a potential risk of sample cracking. When the level
of stress exceeds the strength of the material, structural damage
follows with fracture formation as its most dramatic outcome
[46-47]. Thermo-mechanical effects in cryopreservation have
been extensively studied by Eisenberg et al. [48].
Kelvin GM Brock bank is an owner and employee of Cell &
Tissue Systems. Lia H Campbell, Zhenzhen Chen, and Elizabeth D
Greene are employees of Cell & Tissue Systems.
Disadvantages
Conclusion
Ice-free cryopreservation strategies can be used for
preservation of most cells and tissues. In some cases, particularly
for cells, cryopreservation by freezing may be easier to perform. It
is in natural and engineered tissue cryopreservation that ice-free
methods excel often permitting significantly better preservation
of cells, the extracellular matrix and tissue functions than freezing
preservation methods.
Acknowledgement
This work was supported in part by a US Public Health
Grant from the National Institute of Biomedical Imaging and
Bioengineering, Grant # R43 EB014614, to KGMB. The content is
Table 1: Ice-free cryopreservation of porcine cartilage -metabolic activity
after rewarming.
Days of Culture
0
1
2
3
4
7
55% Cryoprotectants*
22.7±2.8
21.6±1.6
26.9±4.7
28.4±5.4
24.0±3.4
26.2±1.0
83% Cryoprotectants*
80.5±6.8
92.8±5.3
101.6±6.7
106.6±11.3
112.8±13.4
102.4±15.0
Full thickness cartilage plugs were preserved with bone attached and
stored below -135°C. After rewarming the bone was removed and the
cartilage plugs tested immediately and at frequent intervals over a week
of tissue culture
*Results presented as the mean±1 standard error, n=5, all statistical
comparisons between groups were significant at p<0.05. Data expressed
in percent of untreated control values using the resazurin assay
Conflict of Interest
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Citation: Brockbank KGM, Chen Z, Greene ED, Campbell LH (2014) Ice-Free Cryopreservation by Vitrification. MOJ Cell Sci Report 1(2): 00007.
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