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Effect of Cryopreservation Protocols On The Phenotypic Stability of Yeast

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CryoLetters 31 (3), 261-267 (2010)

© CryoLetters, businessoffice@cryoletters.org

EFFECT OF CRYOPRESERVATION PROTOCOLS ON THE


PHENOTYPIC STABILITY OF YEAST
Pushpa Gujjari, Tamara Muldrow and Jianlong Jim Zhou*

*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

The effect of cryopreservation protocols on the long-term viability, phenotypic stability


and genetic integrity of preserved live biomaterials continues to be of great interest as more
microbes, cell lines, tissues, and even organs are desired for stable and indefinite preservation.
Various cryopreservation protocols including vitrification are currently employed for
functional preservation of a variety of biomaterials (11, 13). The viability of most preserved
live biomaterials has been maintained well during long-term preservation and revived
biomaterials largely retain the same phenotype as their prefreezing counterparts have (13).
The genetic integrity of cryopreserved live materials has been less well studied, and was often
perceived not greatly affected during cryopreservation as they are largely kept metabolically
arrested (dehydrated and inactive) during the cryo-process (12). However, this perception has
been challenged by several recent findings. For example, Kopeika et al. (8) reported
increased frequencies of mutations, detected by sequencing, in the mitochondrial DNA of
cryopreservared zebrafish (Danio rerio) blastomeres. Mitochondrial DNA mutants of the
yeast Saccharomyces cerevisiae were obtained after freezing (15) and increased Ty1
transposition frequencies were observed in the same yeast after atypical freezing (14).

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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.

MATERIALS AND METHODS

Yeast strain and reagents


The yeast strain of S. cerevisiae ATCC® 208357 with the genotype MATa trp1-1 top2-1ts
ade2 ura3-1 his3-11 leu2-3,112 was employed for this study. Four cryoprotective agents
(dimethlyl sulphoxide or DMSO [Sigma, Cat. No. D2650], glycerol [Sigma, Cat. No.
G5516], sorbitol [Sigma, Cat. No. S1876], and sucrose [Sigma, Cat. No. S1888]) were
evaluated at the following concentrations: DMSO at 8.0% (~1.0 M), glycerol at 10.0% (~1.0
M), sorbitol at 18.2% (~1.0 M), and sucrose at 18.2% (~0.53 M). Each cryoprotectant was
dissolved in distilled water and then filter-sterilized prior to use.

Frozen sample preparation and freezing conditions


Yeast cells for cryopreservation were prepared as follows: Cells were cultured in YPAD
broth (yeast extract 10 g/L, peptone 20 g/L, glucose 20 g/L, and adenine 100 mg/L) with
gentle rotary shaking (100 rpm) at 25°C. When the culture reached a density of approximately
2.0 OD600 nm, cells were harvested by centrifugation at 500xg for 5 minutes at room
temperature and washed once with sterile water, then suspended in one of the four
cryoprotectant solutions to make a cell suspension at the density of approximately 1.1x108
cells/mL. Cell numbers were estimated according to an empirical formula (one OD600 nm
equals to 3×107 cells/mL). 300 L of such a cell suspension was dispensed into each pre-
labeled 1.2-mL cryogenic vial (Nalgene, Cat. No. EF6835A). The vials, placed in a cardboard
box and then covered, were then temporarily stored at 4°C for two hours before being moved
into a 80°C mechanical freezer for freezing and long-term storage or before being subjected
to preservation in liquid nitrogen. In the latter case, sample vials were transferred to the
chamber of a programmable freezer (CryoMed Freezer 7454, Thermo Electron Corp.) and
were subjected to a freezing program in which the chamber temperature was first decreased to
2°C at 1.0°C /min from room temperature, then quickly dropped to 65°C at the rate of
25°C /min. The chamber was then warmed to 12°C; the vials in the chamber went through a
slow freezing step at the rate of 1.0°C /min to 40°C, and a fast freezing step at the rate of

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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).

Viability and phenotypic stability testing


Three frozen vials for each treatment were retrieved from storage containers at each time
point and thawed slowly at room temperature (2123°C) for 15 minutes. The cell-
cryoprotectant mixture in each vial was then transferred into a round bottom Falcon 14-mL
sterile polypropylene tube. To collect any remaining cells, 0.5mL of sterile distilled water was
dispersed to each vial and the content is then transferred to the same Falcon tube. The content
of three frozen vials of each treatment is combined into the same 14-mL tube at this step. The
cells were then pelleted by centrifugation at 500xg for 5 minutes at room temperature and the
supernatant was removed carefully by gentle pipetting to avoid loss of the cells. The cell
pellet (free of cryoprotectant) in each Falcon tube was added with 3.0 mL YPAD liquid
medium to make cell suspension, which was subsequently incubated at 25°C for two hours
with gentle shaking for cell recovery from freezing stress. The cells were pelleted again under
the same condition and the supernatant was again removed carefully by gentle pipetting.
Cells were finally suspended in an appropriate volume (~1.0 mL) by adding filter-sterilized
dropout base (DOB) (Qbiogene, Cat. No. 4025) buffer solution. 100 L of such a cell
suspension was plated on a desired medium and evenly spread. Replicas were three plates for
each treatment at 0, 6, and 20 months, or five to ten plates at 40 and 55 months.
To minimize any unintentional introduction of variables over the study period, media
were always prepared two days before use at each time point. Media used in this study
include YPAD (rich medium) and chemically defined tryptophan dropout media CSM-Trp
(Qbiogene, Cat. No. 4511 for CSM-Trp [complete supplement mixture minus tryptophan]),
prepared according to vendor’s instructions.
For testing on CSM-Trp medium and for calculating overall viable cell counts of each cell
suspension, serial dilutions of recovered cells were performed and then plated. All the plates
were then incubated at 25°C. During incubation, colonies on each plate were counted at 48
hours and thereafter re-counted every 24 hours for new colonies. Total colony counts were
recorded up to 144 hours of incubation. Each colony is presumably grown from a single cell
spread on the plate. For viability counts, any single plate (Petri dish, diameter = 90 mm) that
had more than 1000 colonies was excluded from the calculations as overcrowding generally
leads to underestimation of colonies on that plate.
Phenotypic reversion rate of the yeast trp1-1 auxotroph is calculated by dividing the
+
number of Trp colonies on the CSMTrp plate by the total number of viable cells plated.
CSMTrp is a medium that lacks the amino acid L-tryptophan, thus serving as an indicator
for testing the ability of a yeast strain to synthesize L-tryptophan for its own growth.

RESULTS AND DISCUSSION

Among the eight cryopreservation protocols (combinations of four cryoprotectants and


two low temperatures) tested, cell viability remained quite steady in seven of them during a
five-year period of storage, as exemplified by the protocol of glycerol as a cryoprotectant and
storage at 80°C (Fig. 1). Viability data on other six protocols (storage in liquid nitrogen with
glycerol, DMSO, sucrose, or sorbitol as a cryoprotectant and storage at 80°C with sucrose
or DMSO as a cryoprotectant) are very similar to those of glycerol at 80°C and thus were

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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. Gly80C and Sor80C 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 Gly80C.

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. DM80C, Gly80C, Sor80C, and Sur80C 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

265
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

Figure 3. Phenotypic reversion frequencies of yeast tryptophan auxotroph to


prototroph (Trp to Trp+) during cryopreservation in liquid nitrogen. T0, T6, T20,
T40, and T55 are the month(s) of 0, 6, 20, 40, and 55 respectively in storage post
initial freezing. DM LN, Gly LN, Sor LN, and Sur LN refer to the preservation in
dimethy sulfoxide, glycerol, sorbitol, and sucrose respectively in liquid nitrogen.
Vertical small bars on the graph are the standard deviation values.

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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Accepted for publication 7/4/2010

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