Biopreservation and Biobanking - Ziath - 15

TOWARD OPTIMAL CRYOPRESERVATION
15
analysis over the numbers of cycles was performed. A
p-value of 0.05 is specified as the significance level.
Results
Influence of temperature cycles on PBMC
viability and recovery
To study the influence of temperature changes during
deep temperature storage, PBMCs from four CMV-positive
donors were isolated and exposed to 0-350 temperature
fluctuations in steps of 50 cycles with a robotic system.
After the simulation of temperature fluctuations during cell
storage, the PBMC viability and recovery were determined
immediately after thawing as well as after overnight culture,
through a trypan blue exclusion test.
The viability of the samples stored without temperature
fluctuations in the vapor phase of LN2 directly after
thawing was 98.33% - 0.65% (Fig. 3A). After 350 temperature
cycles, the viability decreased to 94.03% - 5.65%. The
difference became statistically significant after 200 temperature
fluctuations (N= 36, one-way ANOVA Bonferroni
with p< 0.05, R2 = 0.17051, Z2 = 1.0089). After overnight
culture, the viability of the samples stored in the gas phase
of LN2 was 95.35% - 1.63% and decreased to 93.87% -
1.83% after 350 temperature cycles (Fig. 3B). A significant
difference was observed after 200, 250, and 350 temperature
cycles (N= 36, one-way ANOVA Bonferroni with p< 0.05,
R2 = 0.06666, Z2 = 1.0019).
Like the PBMC viability, the recovery decreased with inFIG.
2. Cycle program in detail. After starting the cycle
program, the sample cabinet is moved into the cold gas
phase above liquid nitrogen until a temperature below
-130C is reached in the reference sample. Next, the sample
cabinet moves up into a room temperature zone until a defined
temperature of -60C is reached in the reference
sample. This process is repeated until the last remaining
samples are exposed to 350 cycles.
Quality assurance
The study was performed in a laboratory, which is under
Good Clinical Laboratory Practice. There are standard operating
procedures and the staff is well trained. The performance
of ELISpot assay is a long-standing process in the
laboratory and is periodically evaluated by an External Quality
Assessment Program Oversight Laboratory (EQAPOL) and
ELISpot Quality Assurance Program.
Statistics
Statistical tests were performed using OriginPro 9.0. All
data are shown as mean - standard deviation. For statistical
testing, we used the individual values. In summary, for
every cycle condition, we performed 36 individual measurements
for viability, 36 for recovery, and triplicates for
T cell functionality. After testing the sample population for
normal distribution through the Kolmogorov-Smirnov test,
statistical differences between storage with and without
temperature fluctuations were calculated by using a one-way
analysis of variance (ANOVA) Bonferroni test. To quantify
the reduction of T cell functionality, a linear regression
creasing numbers of temperature fluctuations, but to a higher
degree. After thawing, the recovery was 96.08% - 6.91% (0
cycles) and decreased to 74.86% - 11.97% after 350 cycles
(Fig. 4A). A significant difference, after thawing, occurred
after 150 temperature cycles (Fig. 4A; N= 36, one-way ANOVA
Bonferroni with p< 0.05, R2 = 0.44609, Z2 = 1.0096).
After an overnight rest, the recovery of samples stored in the
gas phase of LN2 was 81.41% - 6.20% and decreased to
57.74% - 9.94% after 350 cycles (Fig. 4B). After overnight
culture, a significant difference was observed after only 50
temperature fluctuations (Fig. 4B, N= 36, one-way ANOVA
Bonferroni with p < 0.05, R2 = 0.502, Z2 = 1.0076).
Influence of temperature cycles
on T cell functionality
To examine a possible influence of temperature fluctuations
on the T cell functionality, IFN-g ELISpots were
performed. To specify positive T cell responses, the average
number of SFCs/1 · 106 PBMCs was determined from three
replicate wells. Following standardization and validation
issues of ELISpot assay,36 we used the definition of responder
R>4D and R>55, where R is the SFCs/1 · 106
PBMCs for the reagents (CEF and CMV) and D corresponds
to SFCs for background. All samples were positive for
both CEF and CMV. Furthermore, we classified the immune
response of donors in IFN-g ELISpot in four different
groups to compare the reactivity: negative <55 SFCs/1 · 106
PBMCs, low responder <600 SFCs/1 · 106 PBMCs, medium
responder <2000 SFCs/1 · 106 PBMCs, and high responder
>2000 SFCs/1 · 106 PBMCs.
After CEF stimulation, none of four donors were classified
negative, two donors as low responders, one donor as
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