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Maximizing the retention of antigen specific lymphocyte function after cryopreservation.

Author(s): Disis ML, dela Rosa C, Goodell V, Kuan LY, Chang JC, Kuus-Reichel K, Clay TM, Kim Lyerly H, Bhatia S, Ghanekar SA, Maino VC, Maecker HT

Publication: J Immunol Methods, 2006, Vol. 308, Page 13-8

PubMed ID: 16337957 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated how the number of cells per vial, freezing media additive, short-term storage on dry ice, resuspension solution volume and temperature, and the speed and duration of post-thaw centrifugation affected PBMC viability. PBMCs were isolated by ficoll gradient from blood collected from 10 volunteers.  PBMCs were cryopreserved in media containing dextran, human AB serum, fetal bovine serum, or human serum albumin at a concentration of 10, 20 or 30 million cells per vial. Cells were stored on dry ice for 0, 24, 48, or 72 h before storage in liquid nitrogen for 30 days or more. Cells were thawed at 37˚C and washed in 15 or 50 mL pre-warmed media at 4˚C, 25˚C or 37˚C. Cells were concentrated by centrifugation at 1200 or 1500 rpm for 5 or 10 min and then resuspended. Viability was assessed by Trypan Blue.

   Summary of Findings:

   PBMC mean viability was significantly lower when cells were cryopreserved in media containing human AB serum rather than dextran, FBS or HSA (77.9% versus 93.9%, 93.3% and 93.5%, respectively, P<0.0001 all). The authors state that cells cryopreserved in media containing dextran tended to clump, which prevented accurate counting. PBMC viability increased with the temperature of washing media (69.7% at 4˚C, 92.55% at 25˚C, and 95.11% at 37˚C, P=0.001). Storage of cryopreserved PBMCs on dry ice for 0, 24 h, 48 h or 72 h before storage in liquid nitrogen resulted in comparable means for viability (98.2% 98.4%, 96% and 97.3%, respectively). No effect on viability was observed for the volume used for washing (15 cc versus 50 cc), post-wash centrifugation duration (5 min vs 10 min) and speed (1200 rpm versus 1500 rpm), and the number of cells per vial (10, 20 or 30 million). 

   Biospecimens

   • Bodily Fluid - Blood
   • Cell - White Cells

   Preservative Types

   • Frozen

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   Cell count/volume	Light microscopy

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Cell number	10 million cells per vial 20 million cells per vial 30 million cells per vial
   Biospecimen Aliquots and Components	Centrifugation	Multiple durations compared Multiple speeds compared
   Storage	Storage duration	0 h 24 h 48 h 72 h
   Analyte Extraction and Purification	Washing	4˚C media 25˚C media 37˚C media 15 mL 50 mL
   Biospecimen Preservation	Fixative additive/buffer	Human serum albumin Fetal bovine serum Dextran Human AB serum

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2. Study Purpose

   This study compared T-cell response to different antigen that were either fresh and or cryopreserved in different media. PBMCs were isolated by ficoll gradient from blood collected from 40 volunteers.  PBMCs were cryopreserved in media containing dextran, fetal bovine serum (FBS), or human serum albumin (HSA) and aliquoted at a concentration of 20 million cells per vial. Cells were stored in liquid nitrogen, thawed at 37˚C and incubated for five days with tetanus toxoid at 1 LFU/mL, phytohemagglutinin (PHA) at a concentration of 2.5 Ag/mL, or MBP at 1 Ag/mL. Afterward, proliferation was determined by a radioactive thymadine uptake assay.

   Summary of Findings:

   PBMC proliferative response was weakly correlated between fresh and cells cryopreserved in media containing HSA and fresh cells (r=0.352, P=0.03), but was not significantly correlated between fresh cells and those cryopreserved in media containing dextran or FBS.  Stimulation indices in response to MBP, PHA and tetanus toxoid were comparable for fresh PBMCs and those cryopreserved in media containing HSA.

   Biospecimens

   • Bodily Fluid - Blood
   • Cell - White Cells

   Preservative Types

   • Frozen
   • None (Fresh)

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   Cell count/volume	Radioassay

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	None (fresh) Frozen
   Biospecimen Preservation	Fixative additive/buffer	Human serum albumin Fetal bovine serum Dextran

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