Preliminary Report: Evaluation of storage conditions and cryococktails during peripheral blood mononuclear cell cryopreservation
Author(s): Cosentino LM, Corwin W, Baust JM, Diaz-Mayoral N, Cooley H, Shao W, Van Buskirk R, Baust JG
Publication: Cell Preservation Technology, 2007, Vol. 5, Page 189
Review Paper? No
Purpose of Paper
The purpose of this paper was to determine the effects of freezing media and temperature cycling on peripheral blood mononuclear cell (PBMC) viability.
Conclusion of Paper
PBMC stored in CryoStar had increased viability at 1 h post-thaw when compared to PBMC stored in a RPMI based freezing media. However, by 24 h post-thaw no difference in viability was found between specimens stored in the two different freezing media. By 24 h, a significant decline in viability in cycled specimens was noted and was independent of the freezing media used. In the temperature cycled specimens, necrosis was elevated compared to fixed temperature PBMC. Apoptosis was also elevated in cycled specimens stored in either CryoStar or RPMI based media. The addition of caspase inhibitors to CryoStar freezing media did not increase cell viability at 24 h in static temperature specimens, but did increase viability of cycled specimens. In conclusion, temperature cycling as occurs during routine shipping and storage alters PBMC viability, but these effects may be partially attenuated by the addition of caspase inhibitors to the freezing media.
Studies
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Study Purpose
The purpose of this study was to determine the effects of freezing media and temperature cycling on PBMC viability using trypan blue and Vybrant apoptosis assay. PBMC were stored at a constant -150 degrees C or cycled 98 times between -150 and -80 degrees C before storage at -150 degrees C.
Summary of Findings:
Cell viability was found to be significantly higher when quantified using trypan blue than when the Vybrant apoptosis assay was used. PBMC stored in CryoStar had increased viability at 1 h post-thaw when compared to PBMC stored in a RPMI based freezing media. However, by 24 h post-thaw, no difference in viability was found between specimens stored in the two different freezing media. Specimens that were cycled between -80 and -150 degrees C had a slight but non-significant increase in cell viability at 1 h compared to PBMC stored at static temperature, but by 24 h, a significant decline in viability in cycled specimens was noted and was independent of the freezing media used. In the temperature cycled specimens, necrosis was elevated compared to fixed temperature PBMC starting at 1 h, but was most notable at 4 h post-thaw. Apoptosis was also elevated in cycled specimens stored in either CryoStar or RPMI based media particularly at 4 and 8 h post-thaw. The addition of caspase inhibitors to CryoStar freezing media did not increase cell viability at 24 h in static temperature specimens, but did increase viability of cycled specimens. This was attributed to a significant decrease in necrosis at 4 h and apoptosis at 8 h in cycled specimens containing protease inhibitors. In conclusion, temperature cycling as occurs during routine shipping and storage alters PBMC viability, but these effects may be partially attenuated by the addition of caspase inhibitors to the freezing media.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Cell count/volume Light microscopy Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Blood and blood products Peripheral blood mononuclear cells
Storage Storage duration 30 days
Storage Storage temperature -150 degrees C
-80 to -150 degrees C (cycling)
Biospecimen Preservation Fixative additive/buffer Fetal bovine serum with DMSO
Cryostar
Cryostar with caspase inhibitors