Effect of Cryopreservation Protocols On The Phenotypic Stability of Yeast
Effect of Cryopreservation Protocols On The Phenotypic Stability of Yeast
Effect of Cryopreservation Protocols On The Phenotypic Stability of Yeast
© CryoLetters, businessoffice@cryoletters.org
*Mycology Program, American Type Culture Collection (ATCC), 10801 University Blvd,
Manassas, Virginia 20110, USA;
Corresponding author email: jzhou@atcc.org
Abstract
Eight cryopreservation protocols were assessed for their effects on the viability and
phenotypic stability of the yeast Saccharomyces cerevisiae during a five-year study. It is
found that viability and phenotypic features have remained largely unchanged when the yeast
was preserved in glycerol, dimethyl sulphoxide, or sucrose at 80°C or in liquid nitrogen.
When sorbitol was used as a cryoprotectant, yeast cells frozen and stored at 80°C manifested
great decreases in viability after six months in storage and concomitantly large fluctuations in
the rate of the trp1 auxotrophic reversion. This phenotypic reversion was stable passage after
passage. Such a degree of phenotypic fluctuations, however, was not observed for yeast cells
preserved in the same sorbitol solution that went through a controlled freezing program and
were subsequently stored in liquid nitrogen. These results indicate that some combinations of
cryoprotective agent, freezing program, and storage temperature disturb biomaterials more
profoundly during cryopreservation and imply a genetic basis of this phenotypic change.
Keywords: Cryopreservation, cryoprotectant, phenotypic stability, genetic, yeast
INTRODUCTION
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Many extrinsic factors have been shown in affecting viability, the prominent phenotype,
and potentially genetic integrity of the biomaterials under cryopreservation; they include the
type of cryoprotective agents, pre-freezing conditioning, rate of freezing, storage condition,
and rate of thawing (3,4,6,18). Kitamoto et al. (7) reported that longer duration of
preservation is correlated to increases in morphological and physiological alterations. In their
comparative study of cell membrane permeating and non-permeating agents on
cryopreservation of mouse sperms, Sztein et al. (17) found that non-permeating agents such
as raffinose and trehalose are better in maintaining the functions (motility and fertility) of
mouse sperms than does a permeating agent, such as glycerol or dimethyl sulphoxide
(DMSO). Suga et al. (16) demonstrated that sorbitol, a non-permeating cryoprotectant, is a
better agent than glycerol or DMSO for cryopreserving competent cells of Saccharomyce
cerevisiae and Schizosaccharomyces pombe. However, whether sorbitol is better than other
cryoprotectants in maintaining long-term phenotypic stability and genetic integrity of the
yeast remains to be determined.
Here we report an assessment of eight cryopreservation protocols for their effect on the
viability and phenotypic stability of the yeast S. cerevisiae during a five-year study. This yeast
has many mutants suitable for monitoring phenotypic change in a large population.
Specifically, the trp1-1 mutant was chosen as it has a frequency of spontaneous phenotypic
reversion amenable for such a study.
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10°C /min to 80°C. Finally those vials were transferred to a liquid nitrogen tank in the
vapor phase (135°C) for long-term storage.
During storage, cryopreserved yeast cells were undisturbed until being tested. Vials were
removed from storage and tested at different time points T0, T6, T20, T 40, and T55 (0, 6, 20,
40, and 55 months, respectively, after initial freezing).
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omitted to simplify the graph. Variations in colony number among replicas (i.e., plates spread
with the same amount of cell suspension) were usually small, but could be substantial (e.g.,
up to 45% of the average viable cell counts at some time points, raw data, not shown), thus
contributing to the larger standard deviation values at the time immediately after freezing (T0)
and at fifty-five months in cryopreservation (T55). Such variations may be due to occasional
experimental errors and fortunately do not significantly affect the viability trend. On the other
hand, cells preserved in 1.0 M sorbitol and stored in a 80°C freezer appeared equally robust
during the first six months of storage, but their viability showed a nearly 90 percent decline in
the frozen samples stored 20 months and longer (Fig. 1).
14 Gly–80C
Viable cells (x107/mL)
Sor –80C
12
10
8
6
4
2
0
T0 T6 T20 T40 T55
Time in cryopreservation
Figure 1. Yeast cell viability during cryopreservation. T0, T6, T20, T40, and T55
are the month(s) of 0, 6, 20, 40, and 55 respectively in storage post initial
freezing. Gly80C and Sor80C refer to the cryopreservation of yeast cells in
glycerol and sorbitol respectively and storage in a -80oC freezer. Vertical small
bars on the graph are the standard deviation values. At T0, the top and bottom
standard deviation value range is for Gly80C.
Similar phenomena were reported by Baeza et al. (1) who found huge decreases in the
viability of an astaxanthin-producing yeast Xanthophyllomyces dendrorhous immediately
after freezing and again between 24 and 65 months during cryopreservation in 20% glycerol
at 70°C. Presently we are unable to offer an evidence-supported explanation for such
viability decreases. But we are sure that this phenomenon is not likely due to an accident
(such as equipment failure during storage) as yeast cells preserved in other three
cryoprotectants were kept in the same box and in the same freezer, as well as tested at the
same time. We speculate that residual cellular metabolism might have occurred during storage
at 80°C in some combinations of cryoprotectant, freezing rate, thawing rate, and biomaterial
and that cryo-injuries might impact DNA and its repair mechanism more significantly in some
cryopreservation conditions than in others tested.
Injuries to DNA during cryopreservation were reported previously in mammals, fish, and
yeasts. The injuries can be small, like point mutations (15), and large, such as chromosomal
fragmentation (2, 9) and transposon-mediated DNA rearrangement (14). Detection of a
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genetic change can be especially difficult if it is small (e.g., nucleotide substitutions) and it
results in no phenotypic change or a very subtle change. In this study, we attempted to use the
trp1-1 phenotypic reversion (Trp to Trp+ ) as an indicator for possible genetic changes. The
TRP1 gene encodes the enzyme phosphoribosylanthranilate isomerase and the trp1-1 allele
has an point mutation (10), thus producing a truncated, non-functional enzyme in the
tryptophan biosysnthesis pathway.
Under the condition of 1.0 M sorbitol and storage at 80°C, we found that concomitantly
with the viability decrease after initial six months, yeast cells exhibited much greater
fluctuations in the Trp to Trp+ reversion rates (Fig. 2). Compared to the similar rates of Trp
to Trp+ between T0 and T6, which have similar viability levels (Fig.1), the reversion rate at
T20 is only around 20% of those at T0 and T6. However, at T40 and T55, the Trp to Trp+
rates are much higher, at approximately 150%, and 190% of the T0 level, respectively (Fig.
2). Thus, a clearly greater phenotypic instability, reflected by the the Trp to Trp+ reversion
rate, is associated with the significantly reduced viability.
16
14
Trp+ colonies (%)
12
10 DM–80C
Gly –80C
8
Sor –80C
6 Sur –80C
0
T0 T6 T20 T40 T55
Time in cryopreservation
Figure 2. Phenotypic reversion frequencies of yeast tryptophan auxotroph to
prototroph (Trp to Trp+) during cryopreservation at -80oC. T0, T6, T20, T40, and
T55 are the month(s) of 0, 6, 20, 40, and 55 respectively in storage post initial
freezing. DM80C, Gly80C, Sor80C, and Sur80C refer to the preservation in
dimethy sulfoxide, glycerol, sorbitol, and sucrose respectively in a 80oC freezer.
Vertical small bars on the graph are the standard deviation values.
Under seven other conditions that maintained steady levels of post-freezing viability, the
tryptophan auxotrophy phenotype of these preserved yeast cells appeared not greatly affected,
as shown by similar rates of the Trp to Trp+ reversion (Figs. 2 and 3). To test whether the
newly gained Trp+ phenotype is stable, as it would be expected from a genetic change, five
Trp+ colonies from each CSM-Trp plate were randomly selected and streaked on fresh CSM-
Trp medium for testing the phenotypic stability of their Trp to Trp+ reversion (or Trp
suppression) phenotype. All proved to be stable Trp+ colonies, suggesting genetic change is
likely the basis of the new Trp+ phenotype. At the molecular level two different mechanisms
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can lead to the Trp to Trp+ phenotype: reversion and suppression. Reversion refers to a
genetic change that reverse a mutation back to its original nucleotide sequence at that
position, while suppression refers to a genetic change somewhere in the genome that
suppresses the mutant phenotype (e.g., tryptophan auxotroph in this case). It would be
interesting to know which mechanism actually happened in those Trp to Trp+ revertents, an
inquiry might be worth investigation in the future.
10
9
Trp+ colonies (%)
8
7
6
DM LN
Gly LN
5
Sor LN
4
Sur LN
3
2
1
0
T0 T6 T20 T40 T55
Time in cryopreservation
In summary, viability and phenotypic stability remained largely steady and showed no
significant changes when yeast cells went through a controlled freezing program and were
subsequently stored in liquid nitrogen, regardless of cell membrane permeatability of the
cryoprotective agents employed. The results support the notion that cryopreservation in liquid
nitrogen for yeasts is a practice of gold standard. The conventional cryopreservation of yeast
cells at 80°C, compared with the controlled freezing and subsequent preservation in liquid
nitrogen, yielded only slightly lower viability in most cryoprotectants. However, when
sorbitol was used as a cryoprotectant and freezing and storage temperatures are 80°C, this
protocol appeared to cause more intracellular disturbance, leading to great changes in vitality,
phenotypic stability and possible genetic integrity.
Acknowledgements: We thank Ken Jones at ATCC preservation lab for his assistance in
cryopreservation and Sung-Oui Suh, Jan Houseknecht, and Brian Beck for their comments on
and critical reading of the manuscript. This study was supported in part by the federal grant
NCRR-P40-RR016465.
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REFERENCES
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