Temperature fluctuations during deep temperature cryopreservation reduce PBMC recovery, viability and T-cell function.
Author(s): Germann A, Oh YJ, Schmidt T, Schön U, Zimmermann H, von Briesen H
Publication: Cryobiology, 2013, Vol. 67, Page 193-200
PubMed ID: 23850825 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of temperature cycling at room temperature or -80˚C during liquid nitrogen storage on peripheral blood mononuclear cell (PBMC) recovery, viability, and antigen-specific immune response.
Conclusion of Paper
Cycling of PBMC between liquid nitrogen and room temperature resulted in significant declines in PBMC recovery and viability, but similar reductions were not found when cycled between liquid nitrogen and a -80˚C protective hood. The antigen-specific immune response to CMV and CEF peptide pools declined under both storage conditions, but the declines were much greater when cycled between liquid nitrogen and room temperature than liquid nitrogen and -80˚C.
Studies
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Study Purpose
This study investigated the effects of cycling tubes 400 times to room temperature or -80˚C for 5 min during liquid nitrogen storage on PBMC recovery, viability, and antigen-specific immune response. Buffy coats were obtained by density gradient separation over lymphocyte separation medium from 10 donors with cytomegalovirus (CMV). Red blood cells were subsequently lysed using Pharm Lyse. PBMC were aliquoted such that each vial contained 10 x 106 PBMC in 1 mL xeno-free cryomedium IBMT I. PBMCs were frozen in a controlled-freezing container and transferred to liquid nitrogen. Five replicate specimens from each donor were subjected to each of the three storage conditions: stored consistently in liquid nitrogen, cycled 400 times between liquid nitrogen and a protective hood at -80˚C for 5 min (tubes reached -102˚C), or cycled 400 times between the outside environment for 5 min (tubes reached -60˚C) and liquid nitrogen. After cycling, vials were thawed in a 37˚C water bath and then placed in pre-warmed HEPES media supplemented with glutamine and heat-inactivated fetal bovine serum (FBS). Cells were resuspended in medium and viability was determined using the ViCell cell analyzer and the Trypan blue exclusion assay. T-cell functionality was assessed by ELISpot.
Summary of Findings:
Immediately after thawing, recovery and viability were reduced in specimens subjected to 400 cycles between room temperature for 5 min and liquid nitrogen storage compared to specimens stored consistently in liquid nitrogen (89.24% versus 94.34%, P<0.005 and 94.59% versus 97.37%, P<0.005; respectively), but there was no significant effect when cycled between -80˚C and liquid nitrogen. Similarly, PBMC recovery and viability after the cells were allowed to rest were significantly decreased in specimens subjected to 400 cycles between room temperature and liquid nitrogen rather than consistently stored in liquid nitrogen (69.15% versus 82.65%, P<0.005 and 90.89% versus 94.28%, P<0.005; respectively), but no effect of cycling to -80˚C was observed. T-cell responses to CEF peptide pools were 6.54% lower when PBMC were cycled between liquid nitrogen and -80˚C and 29.71% lower when cycled between liquid nitrogen and room temperature than those stored consistently in liquid nitrogen. Similar declines were also observed in T-cell responses to CMV peptide pools in PBMC cycled between liquid nitrogen and -80˚C or room temperature compared to those stored consistently in liquid nitrogen (4.36% and 28.02%, respectively).
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Cytomegalovirus
Platform:
Analyte Technology Platform Cell count/volume Hematology/ auto analyzer Cell count/volume ELISpot Cell count/volume Light microscopy Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Freeze/thaw cycling 1 cycle
400 cycles
Storage Thaw temperature/condition -80˚C
Room temperature