Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Toward Optimal Cryopreservation and Storage for Achievement of High Cell Recovery and Maintenance of Cell Viability and T Cell Functionality.

Author(s): Angel S, von Briesen H, Oh YJ, Baller MK, Zimmermann H, Germann A

Publication: Biopreserv Biobank, 2016, Vol. 14, Page 539-547

PubMed ID: 27792414 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of temperature fluctuations during frozen storage on viability and recovery of PBMCs and on T cell functionality. PBMCs were isolated from buffy coat samples from four healthy cytomegalovirus (CMV)-seropositive donors (collection details not provided) by density gradient centrifugation (details not provided) and aliquots of 1 x 10⁷ PBMCs/mL were frozen at a controlled rate of -1°C/min from +4°C to -80°C and then transferred into vapor-phase LN₂ storage. To simulate specimen handling processes, aliquots were cycled 0-350 times from below -130°C to up to -60°C. This was accomplished using a robotic system that exposed the specimens to room temperature for ~5 minutes until the temperature inside the vial reached a defined temperature of -60°C and then returned them to vapor-phase LN₂ storage. Three specimens were thawed in a water bath after every 50 cycles (0, 50, 100, 150, 200, 250, 300, 350), centrifuged at 400 x g for 6 min at room temperature, resuspended in medium and incubated overnight at 37°C and 5% CO₂. Cell recovery and viability were determined using the trypan blue dye exclusion test and measured with an automated cell analyzer. To determine T cell response, 1 x 10⁵ PBMCs cultured overnight were added to a 96-well plate pre-coated with anti-IFN-γ antibodies and then stimulated by addition of CMV pp65 peptides or CEF peptides (mixture of cytomegalovirus, Epstein-Barr virus, and influenza virus peptides) and incubation for 20–22 hours. Positive T cell response was determined by measurement of spot-forming cells (SFCs per million white cells) by an immunoassay analyzer.

   Summary of Findings:

   Viability of cells decreased significantly after 200 temperature fluctuations and continued to decrease with increasing cycles when compared to specimens not subjected to temperature fluctuations and analyzed immediately after thawing (P<0.05 all). Viability of cells after overnight culture decreased significantly after 200, 250, and 350 temperature cycles (P<0.05). Similarly, recovery of cells decreased with increasing numbers of temperature fluctuations with a significant difference observed after thaw after 150 temperature cycles (P<0.05) and after overnight culture beginning at 50 temperature fluctuations (P<0.05). Linear regression analysis of T cell responses showed decreases with every 50th temperature cycle of cultured cells after stimulation with CEF peptides (P=0.00529) or CMV peptides (P=0.01494) beginning after 50 cycles (-11.39% ± 9.52% after CEF stimulation and -7.38% ± 8.73% after CMV stimulation) and reaching a maximum decrease after 350 cycles (-27.70% ± 13.80% after CEF stimulation and -22.52% ± 11.44% after CMV stimulation).

   Biospecimens

   • Cell - White Cells

   Preservative Types

   • Frozen

   Diagnoses:

   • Cytomegalovirus

   Platform:

   Analyte	Technology Platform
   Protein	Immunoassay
   Cell count/volume	Coulter counter

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Freeze/thaw cycling	0 cycles 50 cycles 100 cycles 150 cycles 200 cycles 250 cycles 300 cycles 350 cycles

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