The idea of
vitrification or achieving a glass-like state was first described in 1860, and
then again in 1937 by Luyet (Luyet,
1937). It wasn’t until
nearly fifty years later in 1985 that Rall and Fahy described vitrification as
a potential alternative to slow-cooling (Rall
and Fahy, 1985). Although
it is relatively successful for embryo storage, it has not allowed
reproducible results for oocytes in any species. Despite this fact, there have been several recent publications
using vitrification to store human oocytes (Ali,
2001; Chen et al., 2000; Chung et
al., 2000; Hong et al., 1999; Hunter et al.,
1995; Kuleshova et al., 1999a;
Kuleshova and Lopata, 2002; Liebermann and Tucker, 2002; Liebermann et
al., 2003; Pensis et al., 1989;
Wininger and Kort, 2002; Wu et al.,
2001; Yoon et al., 2003).
Vitrification involves
exposure of the cell to high concentrations of cryoprotectant(s) for brief
periods of time, usually at or near room temperature, followed by rapid
cooling in liquid nitrogen. The
high osmolarity of the vitrification solution rapidly dehydrates the cell and
submersion into liquid nitrogen quickly solidifies the cell so that the
remaining intracellular water does not have time to form damaging ice
crystals. A similar situation
occurs during slow-cooling, the cells are dehydrated over a longer period of
time and then plunged into liquid nitrogen at much lower temperatures.
Vitrification tries to mimic the extra- and intra-cellular conditions
that exist and allow for survival when cells are plunged (around ‑30°C)
during slow cooling. However, it
may be impossible, at room temperature, to mimic the intracellular environment
that occurs at –30°C, by simply duplicating the extracellular environment
that occurs at –30°C.
Vitrification studies
of the past decade have been among the most interesting in the field of
embryo/oocyte cryopreservation. Because
there is little known about how to vitrify oocytes, and it is different than
slow-cooling (to some extent), investigators have tried many novel approaches.
This has led to several interesting findings.
Many types and combinations of permeable and impermeable
cryoprotectants have been used from propanediol and glycerol, to ficoll and
trehalose. A lot of the methods were borrowed from mouse and bovine
vitrification studies (Martino
et al., 1996; Shaw et al., 1992; Vajta et al.,
1998). Vitrification is a
favorable alternative to slow-cooling in those species that exhibit
sensitivity to a reduction in temperature, including cows and pigs.
Therefore, there have been numerous investigations using these species.
Along with different cryoprotectants, various storage devices have been
used including open-pulled straws, cryo loops, electron microscopy grids, as
well as regular cryo straws (for review see (Liebermann
et al., 2002).
Compared with
slow-freezing, however, vitrification seems to pose a greater threat to
survival because of the toxicity of the highly concentrated cryoprotectants
used and the temperatures at which they are used (Hotamisligil et
al., 1996; Mukaida et al., 1998).
The high-risk involved with vitrification limited the number of reports
using this technique for human oocyte storage until Kuleshova et al.,
(1999) documented the birth of a healthy girl from vitrifying oocytes in
open-pulled straws, a method adopted from a successful bovine vitrification
study (Vajta et
al., 1998). Several
ideas associated with vitrification have since become prevalent in the
literature. These include: 1)
high concentrations of cryoprotectants are toxic and exposure (to the final
and highest concentrations) should be reduced to 30 seconds or less (Martino
et al., 1996; Shaw et al.,
1992); and 2) the faster the cooling rate, the better the survival.
First, toxicity is more of a concern with vitrification because of the
high cryoprotectant concentration (4M to 6M).
The toxic effects of highly concentrated vitrification solutions have
been well established. Most
papers include a pre-equilibration period using a reduced cryoprotectant
concentration, prior to a very short (<30 sec) incubation in the final
vitrification solution (Chung
et al., 2000; Hong et
al., 1999; Hunter et al., 1995;
Shaw et al., 1992; Wu et al., 2001; Yoon et al.,
2003) all based, in part, upon the work of (Martino
et al., 1996). However,
this is not the only method that can work.
By contrast, (Chen
et al., 2000) vitrified human
oocytes in straws, and found that human oocytes could be exposed to the
vitrification solution for either 60 sec or 90 sec prior to plunging in liquid
nitrogen and still survive. Hotamisligil
et al. (Hotamisligil
et al., 1996) reported no significant differences in
development of mouse oocytes incubated in 6M ethylene glycol for 5 min, as
compared to controls. By
contrast, exposure of mouse oocytes to 8M ethylene glycol proved lethal. In a recent article, Isachenko et al. (Isachenko et al.,
2003) showed that human pronuclear oocytes sustained greater damage to
intracellular organelles when cooled without cryoprotectants, therefore
although possibly toxic at high concentrations, cryoprotectants afford
protection. Using a modified
open-pulled straw technique 71% of the zygotes survived and several
pregnancies were reported. Second,
there is the idea that faster cooling rates yield higher survival rates.
This concept is based upon the idea that if the cell is dehydrated to a
certain degree and then cooled fast enough, everything will “freeze” in
place and damage will not have time to occur; crystals will not be able to
organize themselves and a vitrified amorphous, glass-like solid will form.
Similarly, thawing of the vitrified solution, must take place at a
relatively fast rate to prevent crystal organization upon rewarming.
In order to freeze faster several new methods have been used.
The first to come along was the open-pulled straw (Vajta
et al., 1998), which reduced the diameter of a conventional
25cc straw, reducing the amount of liquid that needed to be loaded, and
increasing the vitrification rate. Then
other methods including electron microscope grids and nylon loops that allowed
direct contact with liquid nitrogen and minute volumes were able to increase
the vitirifcation speed considerably (Hong
et al., 1999; Lane and Gardner,
2001; Liebermann et al., 2003;
Martino et al., 1996; Wu et al., 2001). However,
care must be taken to interpret results correctly. If cells die during vitrification it may or may not be
because the cryoprotectant concentration was toxic, or ice formed, or the
cooling rate was too slow. This
is similar to the assumption that IIF and solution effects are the reason that
most cells die during slow-cooling.
With continued success
and pregnancies being reported, vitrification is well on its way to being used
clinically, however there are a few major obstacles that need to be overcome (Kuleshova
and Lopata, 2002). First,
several reports of viral contamination in liquid nitrogen have appeared in the
literature and are cause for concern when not vitrifying in a sealed container
(Bielanski et al., 2003; Bielanski et al.,
2000; Kuleshova and Shaw, 2000). Second,
the common technique of placing a cell or cells into a highly concentrated
vitrification solution, loading them onto a grid, loop, or into a straw, and
plunging, all in less than 30 sec remains technically challenging and more
importantly, leaves little or no room for error, despite what some
investigators say. Third, the
consistency of results with vitrification protocols is often poor.
A survival rate of 70% may be considered good only if it is not the
average of one freeze at 100% and one at 40% (Liebermann
et al., 2002). There
have been reports in the literature that suggest other methods which avoid
these problems are possible. Mouse
oocytes (Wood et
al., 1993) and bovine blastocysts (Kaidi et al.,
2000; Kaidi et al., 1998; Kaidi et
al., 1999) have been successfully vitrified in sealed straws that
have been placed for a period of several minutes in liquid nitrogen vapors
prior to plunging. These reports
show that a longer period of time (enough to load the cells into a straw and
seal the straw, > 1 minute) and a reduced rate of cooling (liquid nitrogen
vapors) can be used and still obtain very good results.
If slower rates of cooling and longer equilibration times are possible,
which these studies demonstrate, the aforementioned two problems could be
avoided (Yokota et
al., 2000; Yokota et al., 2001).
There are many methods
that could work to successfully vitrify cells.
In an early study, Martino suggested that cell dehydration is more
important than having a large amount of cryoprotectant inside the cell (Martino
et al., 1996). The
short equilibration times employed in many vitrification protocols seem to
confirm this idea. This concept
led Kuleshova et al. to add high molecular weight polymers (ficoll or dextran) to the vitrification solution, and thereby reduce the amount of
penetrating cryoprotectants necessary to vitrify oocytes and/or embryos (Kuleshova
et al., 1999b; Kuleshova et al.,
2001). This novel approach
worked well and provides an option for vitrification that reduces the toxicity
of the final solution. If the
toxicity of the vitrification solution is indeed reduced, this may allow for
longer equilibration times which may be necessary to eliminate errors when
trying to vitrify cells in under 30 sec, which many of today’s protocols
employ.
There are several concerns
that may inhibit success in this field. First,
it is a bit disconcerting that investigators using liquid nitrogen vapors for
cooling do not report the temperature of the vapors they are using. Vapor temperature can undergo extreme fluctuations (-20°C to
–170°C) in a matter of centimeters, depending on the width of the
container’s opening used for cooling. Unless
the cooling temperature is standardized (by using a thermocouple, for
example), the rate of cooling/vitirifcation can fluctuate widely and
potentially impact survival rates, or at least be a source of uncontrolled
variance. Second, it appears from
reading both the slow-cooling and vitrification literature that investigators
are concerned about using the quickest, easiest method, instead of first
finding a method that works (90-100% survival), no matter how long and
complicated it may be, and then determining if it can be simplified and/or
shortened.
For more information regarding
cryopreservation or specifically human oocyte cryopreservation see the special
upcoming issue of Reproductive BioMedicine Online (RBMO).
Due out late 2003 or early 2004. And
stay tuned to the Galileo website for monthly updates in the field of
cryopreservation.
