Biopreservation and Biobanking - Ziath - 14

14
ANGEL ET AL.
FIG. 1. Automated robotic system with robotic arm and sample cabinet. Robotic arm changed vertical position. Samples
exposed to room temperature and temperature below -130C. Temperature shift simulates storage conditions during
biobanking. Modified from Germann et al.30
determined by using the trypan blue dye exclusion test. The
measurement was performed using an automated cell analyzer
(ViCell cell analyzer; Beckman Coulter, Krefeld,
Germany). The cell viability and cell recovery from three
samples per cycle condition per donor were measured three
times immediately after thawing as well as after overnight
culture.
%viability ¼
number of viable PBMCs
number of total PBMCs
Viability:
Recovery (%) directly after thawing:
Recovery (%) after overnight culture:
%recovery directly after thawing¼
number of viable PBMCs after thawing
number of frozen viable PBMCs
%recovery after overnight culture¼
· 100
· 100
peptide pool (peptide mixture of cytomegalovirus, EpsteinBarr
virus, influenza virus; CTL, Aalen, Germany), and
PHA (phytohemagglutinin; Thermo Fisher Scientific) as a
positive control. Thawing medium containing 0.4% dimethyl
sulfoxide (DMSO; Sigma-Aldrich) was used as
background control.
After overnight culture, 1 · 105 PBMCs were added into
the wells of a precoated anti-human-IFN-g mAb 1-D1k 96well
plate (Mabtech AB, Hamburg, Germany). To stimulate
an immune response, the different peptides were added to a
final concentration of 2 mg/mL CMV, 4 mg/mL CEF, and
1 mg/mL PHA and incubated at 37C and 5% CO2 for 20-
22 hours. After incubation, the plate was washed with PBSTween
(PBS supplemented with 1% FCS and 250 mL Tween
20; Sigma-Aldrich). The production of IFN-g was detected
by using a biotinylated detection antibody (mAb 7-B6-1;
Mabtech AB) diluted 1:500 in PBS +10% FCS and incubated
for 3 hours and a streptavidin-horseradish peroxidase
complex (Mabtech AB) diluted 1:300 in PBS +10% FCS
and incubated for 1 hour. After the incubation time, the plate
number of viable PBMCs after overnight culture
(number of frozen viable PBMCsnumber of PBMCs removed for measurement directly after thawing)
Determination of T cell functionality
IFN-g ELISpots were performed to determine the influence
of temperature changes during deep temperature storage
on T cell functionality.
The immune response was stimulated by CMV pp65
peptide pool (cytomegalovirus; JTP, Berlin, Germany), CEF
· 100
was washed with PBS-Tween and PBS. The spot development
was performed by using the Nova Red Substrate Kit
(Vector, Burlingame, CA) and stopped after 15 minutes,
washing the plate from every site with distilled water. After
drying the plate overnight, the spots were evaluated with
Immunospot Analyzer (CTL) and the results were specified
as spot-forming cells (SFCs per million PBMCs).

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